Compositions and methods for treating and preventing neointimal stenosis

ABSTRACT

Methods for treating or preventing neointima stenosis are disclosed. The methods generally involve the use of a TGFβ inhibitor, a SMAD2 inhibitor, an FGF Receptor agonist, a Let-7 agonist, or a combination thereof, to inhibit endothelial-to-mesenchymal transition (Endo-MT) of vascular endothelial cells into smooth muscle cells (SMC) at sites of endothelial damage. The disclosed methods can therefore be used to prevent or inhibit neointimal stenosis or restenosis, e.g., after angioplasty, vascular graft, or stent. Also disclosed are methods for increasing the patency of biodegradable, synthetic vascular grafts using a composition that inhibits Endo-MT. A cell-free tissue engineered vascular graft (TEVG) produced by this method is also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of a U.S. patent application Ser. No.14/123,728 filed on Jun. 16, 2014, which is a 371 application of theInternational Application No. PCT/US2012/040759 entitled “Compositionsand Methods for Treating and Preventing Neointimal Stenosis”, filed inthe United States Receiving Office for the PCT on Jun. 4, 2012, whichclaims the benefit of and priority to U.S. Provisional Application No.61/520,040 filed Jun. 3, 2011, U.S. Provisional Application No.61/494,683, filed Jun. 8, 2011, and U.S. Provisional Application No.61/555,712, filed Nov. 4, 2011, which are hereby incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under HL053793,HL098228, and AR046032 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Dec. 3, 2013 as a text file named“YU_5546_5658_5689_ST25.txt,” created on Dec. 3, 2013, and having a sizeof 22,666 bytes is hereby incorporated by reference pursuant to 37C.F.R. §1.52(e)(5).

FIELD OF THE INVENTION

The invention is generally related to the field of vascular stenosis andrestenosis, more particularly to compositions and methods for theprevention and treatment of neointimal hyperplasia or the formation oftissue engineered vascular graft stenosis.

BACKGROUND OF THE INVENTION

Intimal or Neointima formation underlies a number of common vasculardiseases, including vascular graft stenosis and restenosis after anangioplasty or stent. Despite decades of investigations, the origin ofneointima still remains controversial with studies variously pointing tothe role of medial smooth muscle cell (SMC) proliferation (Costa M A andSimon D L Circulation, 111(17):2257 (2005)), vessel wall inflammation(Ohtani K, et al. Circulation. 110(16):2444 (2004)) and adventitialangiogenesis (Khurana R, et al. Circulation, 110(16):2436 (2004)).

Neointimal stenosis followed by thrombosis is the major cause ofsynthetic vascular graft failure. Tissue engineered vascular grafts(TEVGs) offer many advantages to these synthetic grafts, but also havelimitations of their own. TEVGs are typically prepared by seedingautologous cells onto a biodegradable polymeric tubular scaffold. Thescaffold degrades by hydrolysis, ultimately leaving only the livingvessel in the patient.

The methodology of seeding synthetic vascular grafts with autologouscells, however, is still problematic for many reasons. First, itrequires an invasive procedure (biopsy) in addition to the need for asubstantial period of time in order to expand the cells in culture thatlimited its clinical utility. This approach also faces the inherentdifficulty in obtaining healthy autologous cells from diseased donors(Poh, et al., Lancet, 365:2122-24 (2005); Solan, et al., CellTransplant., 14(7):481-8 (2005)). The use of cell culture also resultsin an increased risk of contamination and even the potential fordedifferentiation of the cultured cells. The use of autologous cells toseed the polymeric grafts also limits the off-the-shelf availability oftissue engineered vascular grafts, thereby limiting their overallclinical utility. TEVGs that do not require cell seeding would offermany therapeutic, economic, and safety advantages.

It is an object of the invention to provide compositions and methods fortreating and preventing neointimal stenosis.

It is a further object of the invention to provide methods forincreasing the patency of biodegradable, synthetic vascular grafts withor without using cell seeding.

It is a further object of the invention to provide a cell-free TEVG withimproved patency and reduced graft stenosis.

SUMMARY OF THE INVENTION

The disclosed compositions and methods are based on the discovery thatan endothelial-to mesenchymal transition (Endo-MT) of vascularendothelial cells into smooth muscle cells (SMC) contributes toneointimal stenosis. Endo-MT in blood vessels is initiated bytransforming growth factor β (TGFβ) signaling. In some embodiments, thisTGFβ signaling is through TGFβ receptor I (TGFβRI) and SMAD2. Endo-MT isnormally suppressed by ongoing fibroblast growth factor (FGF) signaling,which maintains Let-7 microRNA expression, which in turn prevents theactivation of TGFβ, TGFβR1 and SMAD2 expression.

A method for treating or preventing neointimal stenosis by inhibiting orreversing Endo-MT is therefore disclosed. The method generally involvesthe use of a composition contains a TGFβ inhibitor, a SMAD2 inhibitor,an FGF receptor agonist, a Let-7 agonist, or a combination thereof toinhibit or prevent Endo-MT in blood vessels with damaged endothelium toprevent or inhibit neointimal stenosis. These compositions may be usedto inhibit or prevent Endo-MT at the site of potential or actualneointima. For example, in some embodiments, the composition is coatedon or incorporated into a vascular device, such as a vascular graft,balloon, or stent prior to administration to a blood vessel of thesubject. In other embodiments, the composition is administered directlyto the subject, e.g., after administration of a vascular device or afterdiagnosis of a neointimal stenosis. In these embodiments, thecomposition is preferably administered locally to the site of actual orpotential neointima formation.

Also disclosed are methods for increasing the patency of biodegradable,synthetic vascular grafts by inhibiting Endo-MT. Cell-free tissueengineered vascular graft (TEVG) produced by this method are alsodisclosed. The methods involve either administering a compositioncontaining a TGFβ inhibitor, a SMAD2 inhibitor, an FGF Receptor agonist,a Let-7 agonist, or a combination thereof either to the subjectreceiving the TEVG or to the TEVG prior to implantation. The TEVGs aretubular, porous structures that allow for recruitment and integration ofhost cells into the graft that mediate remodeling and vascular neotissueformation. The TEVGs are biodegradable, which allows for the grafts tobe completely replaced by forming neotissue as they degrade.

The disclosed TEVGs do not require cell seeding, avoiding many problemsassociated with seeding, including the need for an invasive procedure toobtain autologous cells, the need for a substantial period of time inorder to expand the cells in culture, the inherent difficulty inobtaining healthy autologous cells from diseased donors, and anincreased risk of contamination and the potential for dedifferentiationof the cells. The disclosed cell-free TEVGs therefore have a greateroff-the-shelf availability and increased overall clinical utility.

The cell-free TEVGs may be fabricated from biodegradable polymers usingany known method. In one embodiment, the polymeric vascular grafts arefabricated from woven or non-woven sheets or felts or polymeric fibers.The polymers and fabrication methods are selected to produce vasculargrafts with biomechanical properties, such as initial burst pressure,suture retention strength, elasticity and tensile strength, suitable foruse as vascular conduits. Polymeric woven or non-woven sheets or feltsmay be further treated with polymeric sealants to modify thebiomechanical properties of the graft and/or to control the totalporosity and pore size distribution range of the vascular graft.

A composition containing a TGTGFβ inhibitor, a SMAD2 inhibitor, an FGFReceptor agonist, a Let-7 agonist, or a combination thereof, may beadministered in an effective amount to prevent, inhibit or reducerestenosis, thrombus or aneurysm formation in implanted polymericvascular grafts. The composition may be administered prior to vasculargraft implantation, at the same time as vascular graft implantation,following vascular graft implantation, or any combination thereof. Inone embodiment, the composition is administered either locally orsystemically from a controlled release formulation.

In a preferred embodiment, the composition is administered locally atthe site of graft implantation using a controlled release formulation.In some embodiments, the composition is incorporated into or onto thepolymeric vascular graft which functions as a controlled releaseformulation. The composition may be dispersed evenly throughout thepolymeric vascular graft using any known suitable method. In anotherembodiment, the composition is encapsulated in a second polymeric matrixthat is coated on or incorporated into the polymeric vascular graft. Insome embodiments, the composition is encapsulated into microspheres,nanospheres, microparticles and/or microcapsules, and seeded into theporous vascular graft.

The cell-free TEVGs may be used as venous, arterial or artero-venousconduits for any vascular or cardiovascular surgical application.Exemplary applications include, but are not limited to, congenital heartsurgery, coronary artery bypass surgery, peripheral vascular surgery andangioaccess.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F are bar graphs showing qRT-PCR mRNA expression (normalizedfold expression) of TGFβ1 (FIG. 1A), TGFβRI (FIG. 1B), TGFβRII (FIG.1C), SM α-actin (FIG. 1D), FSP1 (FIG. 1E), and Vimentin (FIG. 1F) incontrol (sold bar) and FRS2 knockdown (shaded bar) HUAEC. *p<0.05;***p<0.001 compared to control.

FIGS. 2A-2I are bar graphs showing qRT-PCR mRNA expression (normalizedfold expression) of SM-calponin (FIGS. 2A, 2D, 2G), Fibronectin (FIGS.2B, 2E, 2H), and Vimentin (FIGS. 2C, 2F, 2I) in ECs treated with theTGFβR1 inhibitor SB431542 (FIGS. 2A-2C), transduced with the dominantnegative TGFβR1 construct TGFβR1 K230R (FIGS. 2D-2F), or treated withSmad2 shRNA (FIGS. 2G-2I). **p<0.01; ***p<0.001; NS: not significantcompared to control.

FIGS. 3A-3B are graphs showing qRT-PCR mRNA expression (relative mRNAlevel) of TGFβRI (FIG. 3A) and Smad2 (FIG. 3B) as a function of time(hr) after 10 μg/ml actinomycin D (ActD) in HUAEC treated with control(-●-) or FRS2 shRNA (-▪-). FIGS. 3C-3D show alignment of let-7 sequenceswith the 3′ITRs of the human. TGFβR1 and Smad2. FIGS. 3E-3J are bargraphs showing qRT-PCR miRNA expression (relative expression) ofhsa-let-7a (FIG. 3E), hsa-let-7b (FIG. 3F), has-let-7c (FIG. 3G),hsa-let-7d (FIG. 3H), hsa-let-7e (FIG. 3I) and let-7a (3J) in HUAECtreated with control (left bars) or FRS2 shRNA (right bars). FIGS. 3K-3Pare plots showing qRT-PCR miRNA expression (relative expression) oflet-7b (FIG. 3K), let-7c (FIG. 3L), let-7d (FIG. 3M), let-7e (FIG. 3N),let-7f (FIG. 3O), and let-7g (FIG. 3P) in E10.5 wild-type (first row),FRS2α+/2F (second row), and FRS2α2F/2F (third row) embryos. Each dot onthe graphs represents one embryo.

FIG. 4A is a graph showing qRT-PCR miRNA expression (relative mRNAlevel) of Lin28 in HUAEC 24 hr (rows 1 and 5), 36 hr (rows 2, and 6), 48hours (rows 3 and 7) or 72 hours (rows 4 and 8) after transduction withan empty vector (rows 1-4) or a vector expressing lin28 (row1s 5-8).FIGS. 4B-4E are bar graphs showing qRT-PCR miRNA expression (relativemRNA level) of hsa-let-7b (FIG. 4B), has-let-7c (FIG. 4C), hsa-let-7i(FIG. 4D), and hsa-mir-98 (FIG. 4E) in HUAEC 24 hr (rows 1 and 5), 36 hr(rows 2, and 6), 48 hours (rows 3 and 7) or 72 hours (rows 4 and 8)after transduction with an empty vector (rows 1-4) or a vectorexpressing lin28 (rows 5-8). FIG. 4F is an illustration of the let-7sponge decoy. FIG. 4G is a bar graphs showing qRT-PCR mRNA expression(relative mRNA level) of TGFβRI (rows 1 and 2), Smad2 (rows 3 and 4),and SM-calponin (rows 5 and 6) in control (sold bars) and lin28overexpressing (shaded bars) HUAEC. FIG. 4H is a bar graphs showingqRT-PCR mRNA expression (relative mRNA level) of TGFβRI (rows 1 and 2),Smad2 (rows 3 and 4), SM-calponin (rows 5 and 6), and HMGA2 (rows 7 and8) in control (solid bars) or let-7 sponge overexpressing (shaded bars)HUAEC. FIGS. 4I-4K are bar graphs showing qRT-PCR mRNA expression(relative mRNA level) of SM α-actin (FIG. 4I), SM22α (FIG. 4J), andSM-calponin (FIG. 4K) in control (rows 1 and 2)) or let-7 spongeoverexpressing (rows 3 and 4) HUAEC untreated (−, rows 1 and 2) ortreated with TGFβ1 (+, rows 2 and 4). FIG. 4L is a bar graph showingqRT-PCR miRNA expression (relative mRNA level) of let-7b (rows 1 and 2)or let-7c (rows 3 and 4) in liver endothelial cells of mice injectedintravenously with PBS (shaded bars) or a single injection of 2 mg/kgcholesterol formulated antagomir-let-7b/c (open bars) that were isolatedat 6 days. n=5 mice in each group. FIG. 4M is a bar graph showingqRT-PCR miRNA expression (relative mRNA level) of let-7a (rows 1 and 2),let-7d (rows 3 and 4), let-7e (rows 5 and 6), let-7f (rows 7 and 8),let-7g (rows 9 and 10), let-7i (rows 11 and 12), miR-98 (rows 13 and 14)in liver endothelial cells of mice injected intravenously with PBS(shaded bars) or a single injection of 2 mg/kg cholesterol formulatedantagomir-let-7b/c (open bars) that were isolated at 6 days. n=5 mice ineach group. FIG. 4N is a bar graph showing qRT-PCR miRNA expression(relative mRNA level) of HMGA2 (rows 1 and 2), CDK6 (rows 3 and 4),CDC25A (rows 5 and 6), TGFβR1 (rows 7 and 8), and Vimentin (rows 9 and10) in liver endothelial cells of mice injected intravenously with PBS(shaded bars) or a single injection of 2 mg/kg cholesterol formulatedantagomir-let-7b/c (open bars) that were isolated at 6 days.

FIG. 5A is a bar graph showing qRT-PCR miRNA expression (relative mRNAlevel) of let-7b (open bars) and let-7c (shaded bars) in HUAEC 0 hrs(rows 1 and 5), 12 hrs (rows 2 and 6), 24 hrs (rows 3 and 7) and 30hours (rows 4 and 8) after transduction with lentivirus vectorexpressing pre-let-7b (open bars) or pre-let-7c (shaded bars). qRT-PCRwas performed for mature let-7 miRNAs. FIGS. 5B-5I are bar graphsshowing qRT-PCR mRNA expression (relative mRNA level) of TGFβRI (FIG.5B), Smad2 (FIG. 5C), FSP1 (FIG. 5D), Fibronectin (FIG. 5E), Vimentin(FIG. 5F), SM α-actin (FIG. 5G), SM22α (FIG. 5H), and SM-calponin (FIG.5I) in control (rows 1 and 2) and FRS2 knockdown (rows 3 and 4) HUAECcells transduced with empty vector (rows 1 and 3) or a lentivirusesexpressing let-7c (rows 2 and 4). *p<0.05; **p<0.01; ***p<0.001 comparedto control.

FIGS. 6A-6G are bar graphs showing qRT-PCR miRNA expression (relativemRNA level) of hsa-let-7a (FIG. 6A), hsa-let-7b (FIG. 6B), hsa-let-7c(FIG. 6C), hsa-let-7d (FIG. 6D), hsa-let-7e (FIG. 6E), hsa-let-7f (FIG.6F), and hsa-let-7g (FIG. 6G) in HUAEC 0 hrs (row 1), 6 hrs (row 2), and9 hrs (row 3) after FGF2 treatment. FIGS. 6H-6M are bar graphs showingqRT-PCR miRNA expression (relative mRNA level) of hsa-let-7b (FIG. 6H),hsa-let-7c (FIG. 6I), hsa-let-7e (FIG. 6J), hsa-let-7f (FIG. 6K),hsa-let-7g (FIG. 6L), and hsa-mir-98 (FIG. 6M) in HUAEC 12 hrs (rows 1and 4), 18 hrs (row 2 and 5), and 30 hrs (row 3 and 6) after treatmentwith DMSO control (shaded bars) or U0126 (solid bars). FIGS. 6N-6Q arebar graphs showing qRT-PCR miRNA expression (relative mRNA level) ofhsa-let-7a (FIG. 6N), hsa-let-7b (FIG. 6O), hsa-let-7c (FIG. 6P), andhsa-let-7e (FIG. 6Q) in control (rows 1 and 2) and FRS2 knockdown (rows3 and 4) HUAEC transduced with an empty vector (−, rows 1 and 3) or witha constitutively active ERK (Ad-ME-LA) (+, rows 2 and 4).

FIGS. 7A-7C are plots showing qRT-PCR mRNA expression (relative mRNAlevel) of TGFβ1 (FIG. 7A), TGFβRI (FIG. 7B), and vimentin (FIG. 7C) inoccluded TEVG at day 7 (row 1) and day 14 (row 2). Each dot on graphrepresents gene expression level in one TEVG; *p<0.01; ***p<0.001compared to control. FIGS. 7D-7E are bar graphs showing TEVG stenosisrate (FIG. 7D) and serum IgG1 (μg/ml) (FIG. 7E) in control (row 1) andsFGFR1-IIIc treated (row 2) mice. Data shown represents mean±SD from 2wells per group.

FIGS. 8A-8B are bar graphs showing the percentage of cells withco-localized LacZ and SM α-actin in patent (row 1) and occluded (row 2)neointimal cells following TEVG insertion in Tie2-Cre; RosaLacZ mice(FIG. 8A) and Cdh5-CreERT2; RosaLacZ mice (FIG. 8B), with the latter Creactivated by tamoxifen administration on day P2. FIGS. 8C-8E are bargraphs showing luminal diameter (mm) (FIG. 8C), stenosis rate (%) (FIG.8D), and percentage of cells with co-localized LacZ and SM α-actin (FIG.8E) in Tie2-CreXRosa26 mice treated with DMSO (row 1) or the TGFβR1inhibitor SB431542 (row 2) after TEVG implantation. Data are presentedas mean±SD; *p<0.05; **p<0.01; ***p<0.001.

FIGS. 9A-9J are bar graphs showing qRT-PCR mRNA expression (relativemRNA level) of SM α-actin (FIG. 9A), SM22a (FIG. 9B), SM-calponin (FIG.9C), SM-MHC (FIG. 9D), Myocardin (FIG. 9E), PDGFRα (FIG. 9F), PDGFRβ(FIG. 9G), Vimentin (FIG. 9H), NG2 (FIG. 9I), and Runx1 (FIG. 9J) inprimary mouse endothelial cells transduced with adenovirus-GFP (soldbars) or adenovirus-Cre (shaded bars) at 4 days. *p<0.05; **p<0.01;***p<0.001.

FIGS. 10A-10F are bar graphs showing qRT-PCR mRNA expression (relativemRNA level) of TGFβRI (FIG. 10A), TGFβRII (FIG. 10B), SM α-actin (FIG.10C), SM22α (FIG. 10D), SM-calponin (FIG. 10E), and Fibronectin (FIG.10F) in control (row 1) or FRF2 2F (row 2), FRS2 4F (row 3), FRS2 6F(row 3), FGFR1 dominant negative (row 4), FGFR1 K562E (row 5), or FGFR1K562EVT (row 6) mutant overexpressing HUAEC.

FIGS. 11A-11I are bar graphs showing qRT-PCR mRNA expression (relativemRNA level) of TGFβR1 (FIG. 11A), TGFβRII (FIG. 11B), Smad2 (FIG. 11C),SM α-actin (FIG. 11D), Fibronectin (FIG. 11E), Vimentin (FIG. 11F),N-cadherin (FIG. 11G), ZEB1 (FIG. 11H), or ZEB2 (FIG. 11I) in control(solid bars) and FRS2 knockdown (shaded bars) primary human mammaryepithelial cells. FIGS. 11J-11M are bar graphs showing qRT-PCR miRNAexpression (relative mRNA level) of hsa-mir-200b (FIG. 11J),hsa-mir-200c (FIG. 11K), hsa-let-7b (FIG. 11L), hsa-let7c (FIG. 11M) incontrol (solid bars) and FRS2 knockdown (shaded bars) primary humanmammary epithelial cells.

FIGS. 12A-12D are bar graphs showing qRT-PCR mRNA expression (relativemRNA level) of TGFβR1 (FIG. 12A), Smad2 (FIG. 12B), COL1A1 (FIG. 12C),and SM α-actin (FIG. 12D) in control (solid bars) and FRS2 knockdown(shaded bars) foreskin cells. FIGS. 12E-12H are bar graphs showingqRT-PCR mRNA expression (relative mRNA level) of TGFβR1 (FIG. 12E),Smad2 (FIG. 12F), COL1A1 (FIG. 12G), and Vimentin (FIG. 12H) in control(solid bars) and FRS2 knockdown (shaded bars) atrial cells.

FIG. 13 is an illustration of the FGF/TGFβ signaling pathway involved inendothelial-to-mesenchymal (Endo-MT) transition. TGFβ activity throughTGFβRI promotes Endo-MT via SMAD2-dependent (smooth muscle cells) andSMAD2-indendent (fibroblasts) pathways. TGFβR1 and SMAD2 are inhibitedby let-7, which is activated by FGF signaling through FGFR. As let-7decreases, TGFβ activity increases and Endo-MT.

FIGS. 14A to 14B are graphs showing absolute (FIG. 14A) and fractional(FIG. 14B) release of SB-431542 from (▪) tethered PLGA microparticlesand (▴) PCLA phase of a PGA-PCLA TEVG scaffold. FIGS. 14C and 14D arebar graphs showing luminal diameter (mm) (FIG. 14C) and stenosis rate(%) of control microparticles (FIG. 14A, bar 1; FIG. 14B, bar 3),SB-431542 microparticles (FIG. 14A, bar 2; FIG. 14B, bar 4), controlscaffold (FIG. 14D, bar 1), and SB-431542 scaffold (FIG. 14D, bar 2).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “vascular stenosis” refers to an abnormal narrowing in a bloodvessel that occurs following an injury to the vessel wall (endothelium).In some embodiments, stenosis involves a reduction in the circumferenceof a lumen of 50% or more. The term “restenosis” refers to stenosis at apreviously stenotic site. Restenosis, as used herein, encompassesocclusion. Exemplary injuries that result in stenosis or restenosisinclude trauma to an atherosclerotic lesion (as seen with angioplasty orstent), a resection of a lesion (as seen with endarterectomy), anexternal trauma (e.g., a cross-clamping injury), or a surgicalanastomosis.

The term “neointimal stenosis” refers abnormal narrowing in a bloodvessel resulting from neointimal formation.

The term “neointima” refers to a new or thickened layer of intima (innerlining) formed in a blood vessel in response to signals from injuredendothelial cells.

The term “TGFβ inhibitor” as used herein refers to a composition thatdirectly inhibits TGFβ or TGFβRI expression, TGFβ or TGFβRIbioavailability, binding of TGFβ to TGFβRI, or the binding of TGFβRI toSMAD2.

The term “SMAD2 inhibitor” as used herein refers to a composition thatdirectly inhibits an activity of SMAD2, such as the phosphorylation ofSMAD2 protein by TGFβRI, the translocation of SMAD2 to the nucleus, orthe binding of SMAD2 to a DNA substrate.

The term “FGF receptor agonist” refers to a composition that directlypromotes FGF signaling through an FGF receptor (FGFR). The agonist maybe an FGF protein (e.g., natural or recombinant FGF), an expressionvector encoding an FGF protein, or any fragment or variant thereof thatactivates FGFR signaling.

The term “Let-7 agonist” refers to a composition that promotes one ormore activities of a Let-7 microRNA. The agonist may be a Let-7 miRNA oran expression vector encoding one or more Let-7 microRNA.

The term “vascular device” refers to a medical device administered to ablood vessel during a medical procedure. The term covers devices thatmay damage the vascular wall and cause neointimal stenosis. The termincludes vascular grafts, angioplasty balloons, and vascular stents.

The term “vector” refers to a replicon, such as a plasmid, phage, orcosmid, into which another DNA segment may be inserted so as to bringabout the replication of the inserted segment. The vectors can beexpression vectors.

The term “expression vector” refers to a vector containing a geneconstruct in a form suitable for expression by a cell (e.g., operablylinked to a transcriptional control element).

The term “operatively linked to” refers to the functional relationshipof a nucleic acid with another nucleic acid sequence. Promoters,enhancers, transcriptional and translational stop sites, and othersignal sequences are examples of nucleic acid sequences operativelylinked to other sequences. For example, operative linkage of DNA to atranscriptional control element refers to the physical and functionalrelationship between the DNA and promoter such that the transcription ofsuch DNA is initiated from the promoter by an RNA polymerase thatspecifically recognizes, binds to and transcribes the DNA.

The term “individual,” “host,” “subject,” and “patient” are usedinterchangeably to refer to any individual who is the target ofadministration or treatment. The subject can be a vertebrate, forexample, a mammal. Thus, the subject can be a human or veterinarypatient.

The term “treat” or “treatment” refers to the medical management of apatient with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

The term “prevent” does not require absolute forestalling of thecondition or disease but can also include a reduction in the onset orseverity of the disease or condition. Thus, if a therapy can treat adisease in a subject having symptoms of the disease, it can also preventthat disease in a subject who has yet to suffer some or all of thesymptoms.

The term “inhibit” refers to a decrease in an activity, response,condition, disease, or other biological parameter. This can include butis not limited to the complete ablation of the activity, response,condition, or disease. This may also include, for example, a 10%reduction in the activity, response, condition, or disease as comparedto the native or control level. Thus, the reduction can be a 10, 20, 30,40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between ascompared to native or control levels.

The term “therapeutically effective” means that the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses or symptoms of a disease or disorder. Such amelioration onlyrequires a reduction or alteration, not necessarily elimination.

The term “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problems or complications commensurate witha reasonable benefit/risk ratio.

The term “small molecule” refers to a molecule, such as an organic ororganometallic compound, with a molecular weight of less than 2,000Daltons, more preferably less than 1,500 Daltons, most preferably lessthan 1,000 Daltons. The small molecule can be a hydrophilic,hydrophobic, or amphiphilic compound.

The term “antibodies” refers to natural or synthetic antibodies thatselectively bind a target antigen. The term includes polyclonal andmonoclonal antibodies. In addition to intact immunoglobulin molecules,also included in the term “antibodies” are fragments or polymers ofthose immunoglobulin molecules, and human or humanized versions ofimmunoglobulin molecules that selectively bind the target antigen.

The term “copolymer” refers to a single polymeric material that iscomprised of two or more different monomers. The copolymer can be of anyform, such as random, block, graft, etc. The copolymers can have anyend-group, including capped or acid end groups.

The term “biocompatible” refers to a material and any metabolites ordegradation products thereof that are generally non-toxic to therecipient and do not cause any significant adverse effects to thesubject.

The term “biodegradable” refers to a material that will degrade or erodeunder physiologic conditions to smaller units or chemical species thatare capable of being metabolized, eliminated, or excreted by thesubject.

The term “controlled release” or “modified release” refers to a releaseprofile in which the active agent release characteristics of time courseand/or location are chosen to accomplish therapeutic or convenienceobjectives not offered by conventional dosage forms such as solutions,suspensions, or promptly dissolving dosage forms. Delayed release,extended release, and pulsatile release and their combinations areexamples of modified release.

The term “bioactive agent” or “active agent” refers to therapeutic,prophylactic, and/or diagnostic agents. It includes without limitationphysiologically or pharmacologically active substances that act locallyor systemically in the body. A biologically active agent is a substanceused for, for example, the treatment, prevention, diagnosis, cure, ormitigation of disease or disorder, a substance which affects thestructure or function of the body, or pro-drugs, which becomebiologically active or more active after they have been placed in apredetermined physiological environment. Bioactive agents includebiologically, physiologically, or pharmacologically active substancesthat act locally or systemically in the human or animal body. Examplescan include, but are not limited to, small-molecule drugs, peptides,proteins, antibodies, sugars, polysaccharides, nucleotides,oligonucleotides, aptamers, siRNA, nucleic acids, and combinationsthereof. “Bioactive agent” includes a single agent or a plurality ofbioactive agents including, for example, combinations of two or morebioactive agents.

II. Compositions

A. TGFβ and SMAD2 Inhibitors

As discussed above endothelial-to-mesenchymal transition (Endo-MT) isinitiated in blood vessels after angioplasty, vascular graft, or otherinjury, by TGFβ signaling. In some embodiments, this TGFβ signaling isTGFβRI- and SMAD2-dependent. Therefore, TGFβ inhibitors and SMAD2inhibitors are disclosed for inhibiting, preventing, and/or reversingEndo-MT in blood vessels.

1. SB431542

In some embodiments, the TFGβ or SMAD2 inhibitor is4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide(SB431542).

SB431542 inhibits the activity of transforming growth factor β1 (TGF-β1)activin receptor-like kinases (ALKs). It is a selective and potentinhibitor of the phylogenetically related subset of ALK-4 (activin typeI receptor), ALK-5 (TGFβRI), and ALK-7 (nodal type I receptor). SB431542inhibits endogenous activin and TGF-β signaling but is without effect onthe more divergent ALK-1, -2, -3, and -6 and hence BMP signaling.Phosphorylation of Smad2 by ectopically expressed constitutively activeALK-4, ALK-5, and ALK-7 in transfected NIH 3T3 cells is completelyabolished by SB431542 at 10 mM (Watabe, T., et al., J. Cell Biol., 163,1303 (2003)). In addition, the compound inhibits ligand dependentactivation of wild type ALK-4 and endogenous ALK-5 with an IC₅₀ ofapproximately 0.25 mM (Watabe, T., et al., J. Cell Biol., 163, 1303(2003)).

2. SB208

In some embodiments, the TFGβ or SMAD2 inhibitor is2-(5-Chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine (SB208). SB208is a potent, orally active, ATP-competitive TGF-βRI inhibitor (IC₅₀=49nM).

3. SB525334

In some embodiments, the TFGβ or SMAD2 inhibitor is6-[2-(1,1-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline(SB525334). SB525334 is a selective inhibitor of TGF-βRI (IC₅₀=14.3 nM).It inhibits TGF-β1-induced smad2/3 nuclear localization andTGF-β1-induced mRNA expression.

4. LY2157299

In some embodiments, the TFGβ or SMAD2 inhibitor is4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide(LY2157299). LY2157299 is a selective kinase inhibitor for TGF-βR1 withIC₅₀ of 86 nM. LY2157299 is well tolerated at doses of 40 mg and 80 mg.

5. EW-7203

In some embodiments, the TFGβ or SMAD2 inhibitor is3((5-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-4-(6-methylpyridin-2-yl)thiazol-2-ylamino)methyl)benzonitrile(EW-7203). EW-7203 inhibits TGF-β1-induced Smad signaling and Endo-MT inmammary epithelial cells (Park C Y, et al. Cancer Sci. 102(10):1889-96(2011)).

6. SM16

In some embodiments, the TFGβ or SMAD2 inhibitor is4-(5-(benzo[d][1,3]dioxol-5-yl)-4-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)bicyclo[2.2.2]octane-1-carboxamide(SM16). SM16 is a 430.5 MW ALK5/ALK4 kinase inhibitor that blocks TGF-βand activin-induced Smad2/3 phosphorylation.

7. Losartan

In some embodiments, the TFGβ inhibitor is(2-butyl-4-chloro-1-{[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-1H-imidazol-5-yl)methanol(Losartan). Losartan (rINN) is an angiotensin II receptor antagonistdrug used mainly to treat high blood pressure that has also been shownto downregulate the expression of TGF-β types I and II receptors.

8. Competitive Binding Molecules

The SMAD pathway is the canonical signaling pathway that TGF-β familymembers signal through. In this pathway, TGF-β dimers bind to a type IIreceptor which recruits and phosphorylates a type I receptor. The type Ireceptor then recruits and phosphorylates a receptor regulated SMAD(R-SMAD).

Therefore, TGFβ signaling can also be inhibited with molecules thatcompetitively bind TGFβ or TGFβRII and inhibit TGFβ signaling. SMAD2activity can also be inhibited with molecules that competitively bindTGFβRI or SMAD2 and inhibit SMAD2 phosphorylation, translocation, orbinding to DNA substrates. Examples of such competitive bindingmolecules as antibodies, soluble receptors (ligand trap), and mutantligands.

Antibodies that can be used in the disclosed compositions and methodsinclude whole immunoglobulin (i.e., an intact antibody) of any class,fragments thereof, and proteins containing at least the antigen bindingvariable domain of an antibody. The variable domains differ in sequenceamong antibodies and are used in the binding and specificity of eachparticular antibody for its particular antigen. The variability is notusually evenly distributed through the variable domains of antibodies.It is typically concentrated in three segments called complementaritydetermining regions (CDRs) or hypervariable regions both in the lightchain and the heavy chain variable domains. The more highly conservedportions of the variable domains are called the framework (FR). Thevariable domains of native heavy and light chains each comprise four FRregions, largely adopting a beta-sheet configuration, connected by threeCDRs, which form loops connecting, and in some cases forming part of thebeta-sheet structure. The CDRs in each chain are held together in closeproximity by the FR regions and, with the CDRs from the other chain,contribute to the formation of the antigen binding site of antibodies.Therefore, the disclosed antibodies contain at least the CDRs necessaryto bind TGFβ, TGFβRI, TGFβRII, or SMAD2 and inhibit TGFβ signaling.

Techniques can also be adapted for the production of single-chainantibodies that bind TGFβ, TGFβRI, TGFβRII, or SMAD2 and inhibit TGFβsignaling. Methods for the production of single-chain antibodies arewell known to those of skill in the art. A single chain antibody can becreated by fusing together the variable domains of the heavy and lightchains using a short peptide linker, thereby reconstituting an antigenbinding site on a single molecule. Single-chain antibody variablefragments (scFvs) in which the C-terminus of one variable domain istethered to the N-terminus of the other variable domain via a 15 to 25amino acid peptide or linker have been developed without significantlydisrupting antigen binding or specificity of the binding. The linker ischosen to permit the heavy chain and light chain to bind together intheir proper conformational orientation. Divalent single-chain variablefragments (di-scFvs) can be engineered by linking two scFvs. This can bedone by producing a single peptide chain with two VH and two VL regions,yielding tandem scFvs. ScFvs can also be designed with linker peptidesthat are too short for the two variable regions to fold together (aboutfive amino acids), forcing scFvs to dimerize. This type is known asdiabodies. Diabodies have been shown to have dissociation constants upto 40-fold lower than corresponding scFvs, meaning that they have a muchhigher affinity to their target. Still shorter linkers (one or two aminoacids) lead to the formation of trimers (triabodies or tribodies).Tetrabodies have also been produced. They exhibit an even higheraffinity to their targets than diabodies.

Soluble TGFβ receptors, such as a soluble TGFβRII receptor, may also beused to bind free TGFβ and prevent activation of TGFβ signaling. SolubleTGFβRI receptor may also be used to bind free SMAD2 and preventphosphorylation of SMAD2. Soluble receptors are generally chimericfusion proteins containing the extracellular domain of the receptorfused to the Fc portion of an immunoglobulin.

A mutant TGFβ ligands that bind TGFβRII without activating receptorsignaling may be used to compete with endogenous TGFβ and inhibit TGFβRIsignaling. A mutant SMAD2 that binds TGFβRI without being phosphorylatedand activating downstream targets may be used to compete with endogenousTGFβRI for binding to endogenous SMAD2.

B. FGF Receptor Agonists

FGF signaling suppresses Endo-MT. Therefore, FGF agonists are disclosedfor use in the disclosed compositions and methods. Therefore, FGFReceptor agonists are disclosed for use in the disclosed compositionsand methods. Any FGF receptor agonists, i.e., agents composition thatdirectly promotes FGF signaling through an FGF receptor (FGFR) can beused in the disclosed compositions and methods.

In some embodiments, the FGF Receptor agonist is natural, synthetic, orrecombinant FGF peptide or protein that can bind and activate FGFReceptor signaling in an endothelial cell, such as a human endothelialcell. The FGF protein is preferably human. Recombinant human FGF-2proteins are commercially available from, for example, INVITROGEN (GrandIsland, N.Y.) and R&D SYSTEMS (Minneapolis, Minn.). Other examples ofFGF proteins which are FGF agonists include FGF1, FGF4 and FGF-5,however, over 20 FGF family proteins (FGF 1-14 and FGF 1-23) have beenidentified, most of which bind to one or more of the 4 FGF receptors.Reviewed in Itoh, et al., Trends in Genetics, 20(10:563-9 (2004).

Expression vectors encoding an FGF protein, or fragment thereof, thatbinds and activates FGF Receptor may also be used. The expression vectormay contain a nucleic acid encoding an FGF agonist operably linked to anexpression control sequence.

Small molecule FGF Receptor agonist that may be used in the disclosedcompositions and methods are described in U.S. Patent Publication No.2009/0069368 by Bono, et al. Other FGF Receptor agonists includeagonistic antibodies specific for FGF receptors, described for examplein Malavaud, et al., Oncogene, 23:6769-6778 (2004).

C. Let-7 MicroRNA Agonists

FGF signaling suppresses Endo-MT by maintaining Let-7 microRNAexpression, which prevents the activation of TGFβ, TGFβR1 and SMAD2.Therefore, Let-7 agonists are disclosed for use in the disclosedcompositions and methods.

In preferred embodiments, the Let-7 agonist is a Let-7 miRNA or apolynucleotide encoding a Let-7 miRNA. In some embodiments, the Let-7agonist is a pri-miRNA, pre-miRNA, mature miRNA, or a fragment orvariant thereof effective in gene silencing, e.g., silencing TGFBRI geneexpression. In other embodiments, the let-7 miRNA is an RNAinterference-inducing (RNAi) molecule including, but not limited to, asiRNA, dsRNA, stRNA, shRNA, or gene silencing variants thereof. Inalternative embodiments the let-7 miRNA is an agent which binds andinhibits an RNA transcript comprising a let-7 target sequence. Examplesof such agents include, but are not limited to a small molecule,protein, antibody, aptamer, ribozyme, nucleic acid or nucleic acidanalogue.

In some embodiments, the Let-7 miRNA is human let-7a1, let-7a2, let-7a4,let-7b, let-7e, let-7d, let-7d, let-7f1, let-7f2, let-7g, let-7i, ormir-98. Of these let-7 family members, Let-7b and Let-7c have the bestsequence match with TGFβR1. The following are sequences for the humanlet-7 family members. Also disclosed are conservative variants of thesesequences having 1, 2, 3, 4, 5, or 6 substitutions, deletions, orinsertions

let- UGGGAUGAGG UAGUAGGUUG SEQ ID 7a1 UAUAGUUUUA GGGUCACACC NO: 102CACCACUGGG AGAUAACUAU ACAAUCUACU GUCUUUCCUA let- AGGUUGAGGU AGUAGGUUGUSEQ ID 7a2 AUAGUUUAGA AUUACAUCAA NO: 103 GGGAGAUAAC UAUACAACCUCCUAGCUUUC CU let- GGGUGAGGUA GUAGGUUGUA SEQ ID 7a3UAGUUUGGGG CUCUGCCCUG NO: 104 CUAUGGGAUA ACUAUACAAU CUACUGUCUU UCCU 1et-CGGGGUGAGG UAGUAGGUUG SEQ ID 7b UGUGGUUUCA GGGCAGUGAU NO: 105GUUGCCCCUC GGAAGAUAAC UAUACAACCU ACUGCCUUCC CUG let-GCAUCCGGGU UGAGGUAGUA SEQ ID 7c GGUUGUAUGG UUUAGAGUUA NO: 106CACCCUGGGA GUUAACUGUA CAACCUUCUA GCUUUCCUUG GAGC let-CCUAGGAAGA GGUAGUAGGU SEQ ID 7d UGCAUAGUUU UAGGGCAGGG NO: 107AUUUUGCCCA CAAGGAGGUA ACUAUACGAC CUGCUGCCUU UCUUAGG let-CCCGGGCUGA GGUAGGAGGU SEQ ID 7e UGUAUAGUUG AGGAGGACAC NO: 108CCAAGGAGAU CACUAUACGG CCUCCUAGCU UUCCCCAGG let- UCAGAGUGAG GUAGUAGAUUSEQ ID 7f1 GUAUAGUUGU GGGGUAGUGA NO: 109 UUUUACCCUG UUCAGGAGAUAACUAUACAA UCUAUUGCCU UCCCUGA let- UGUGGGAUGA GGUAGUAGAU SEQ ID 7f2UGUAUAGUUU UAGGGUCAUA NO: 110 CCCCAUCUUG GAGAUAACUAUACAGUCUAC UGUCUUUCCC ACG let- AGGCUGAGGU AGUAGUUUGU SEQ ID 7gACAGUUUGAG GGUCUAUGAU NO: 111 ACCACCCGGU ACAGGAGAUAACUGUACAGG CCACUGCCUU GCCA let- CUGGCUGAGG UAGUAGUUUG SEQ ID 7iUGCUGUUGGU CGGGUUGUGA NO: 112 CAUUGCCCGC UGUGGAGAUAACUGCGCAAG CUACUGCCUU GCUA mir- AGGAUUCUGC UCAUGCCAGG SEQ ID 98GUGAGGUAGU AAGUUGUAUU NO: 113 GUUGUGGGGU AGGGAUAUUAGGCCCCAAUU AGAAGAUAAC UAUACAACUU ACUACUUUCC CUGGUGUGUG GCAUAUUCA

In some embodiments, the Let-7 agonist is a vector encoding one or morelet-7 miRNA operably linked to a transcription control element. Forexample, genes inserted in viral and retroviral systems usually containpromoters and/or enhancers to help control the expression of the desiredgene product. A promoter is generally a sequence or sequences of DNAthat function when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required forbasic interaction of RNA polymerase and transcription factors, and maycontain upstream elements and response elements. Enhancer generallyrefers to a sequence of DNA that function at no fixed distance from thetranscription start site and can be either 5′ or to the transcriptionunit. Furthermore, enhancers can be within an intron as well as withinthe coding sequence itself. They are usually between 10 and 300 bp inlength, and they function in cis. Enhancers function to increasetranscription from nearby promoters.

In some embodiments, the promoter is inducible. In other embodiments,the promoter is a constitutive to maximize expression of the region ofthe transcription unit to be transcribed. In some embodiments, thepromoter is tissue-specific, e.g., expressed only in vascularendothelial cells.

D. Pharmaceutical Compositions

Pharmaceutical compositions containing therapeutically effective amountsof a TGFβ inhibitor, a SMAD2 inhibitor, an FGF Receptor agonist, a Let-7agonist, or a combination thereof optionally in a pharmaceuticallyacceptable carrier/excipient are disclosed.

1. Formulations for Parenteral Administration

In one embodiment, the pharmaceutical composition is administered in anaqueous solution by parenteral injection. The formulation may also be inthe form of a suspension or emulsion. In general, pharmaceuticalcompositions are provided including effective amounts of a TGFβinhibitor, a SMAD2 inhibitor, an FGF Receptor agonist, a Let-7 agonist,or a combination thereof, and optionally include pharmaceuticallyacceptable diluents, preservatives, solubilizers, emulsifiers, adjuvantsand/or carriers. Such compositions include diluents sterile water,buffered saline of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength; and optionally, additives such asdetergents and solubilizing agents (e.g., TWEEN® 20, TWEEN® 80,Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol). Examples of non-aqueoussolvents or vehicles are propylene glycol, polyethylene glycol,vegetable oils, such as olive oil and corn oil, gelatin, and injectableorganic esters such as ethyl oleate. The formulations may be lyophilizedand redissolved/resuspended immediately before use. The formulation maybe sterilized by, for example, filtration through a bacteria retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions.

2. Controlled Delivery Polymeric Matrices

In one embodiment, the pharmaceutical composition containing a TGFTGFβinhibitor, a SMAD2 inhibitor, an FGF Receptor agonist, a Let-7 agonist,or a combination thereof is administered systemically or locally usingcontrolled release formulations. In a preferred embodiment, thepharmaceutical composition is administered locally at the site of avascular device using a controlled release formulation.

In one embodiment, the pharmaceutical composition is coated on orincorporated into a polymeric vascular graft which functions as acontrolled release formulation. The pharmaceutical composition may bedispersed throughout the polymeric vascular graft using any knownsuitable method. For example, the pharmaceutical composition is may beadded to the polymeric scaffold during fabrication by adding it to thepolymer solution or emulsion or during the fabrication of a polymerictextile, such as by an electrospinning process. Additionally, oralternatively, the pharmaceutical composition may be added to thepolymeric graft following fabrication. In some embodiments, thepharmaceutical composition is localized to either the exterior or thelumen of the tubular polymeric vascular graft.

In another embodiment, the pharmaceutical composition may beencapsulated in a polymeric matrix that is either administered to thesite of actual or potential neointimal stenosis or incorporated into thepolymeric vascular graft. In some embodiments, the pharmaceuticalcomposition is encapsulated into microspheres, nanospheres,microparticles and/or microcapsules. The matrix can be in the form ofmicroparticles such as microspheres, where the active agent is dispersedwithin a solid polymeric matrix or microcapsules, where the core is of adifferent material than the polymeric shell, and the active agent isdispersed or suspended in the core, which may be liquid or solid innature. Unless specifically defined herein, microparticles,microspheres, and microcapsules are used interchangeably. Alternatively,the polymer may be cast as a thin slab or film, ranging from nanometersto four centimeters, a powder produced by grinding or other standardtechniques, or even a gel such as a hydrogel, and used as a coating forthe vascular graft.

Either non-biodegradable or biodegradable matrices can be used fordelivery of the pharmaceutical composition, although biodegradablematrices are preferred. These may be natural or synthetic polymers,although synthetic polymers are preferred due to the bettercharacterization of degradation and release profiles. The polymer isselected based on the period over which release is desired. In somecases linear release may be most useful, although in others a pulserelease or “bulk release” may provide more effective results. Thepolymer may be in the form of a hydrogel (typically in absorbing up toabout 90% by weight of water), and can optionally be crosslinked withmultivalent ions or polymers.

The matrices can be formed by solvent evaporation, spray drying, solventextraction and other methods known to those skilled in the art.Bioerodible microspheres can be prepared using any of the methodsdeveloped for making microspheres for drug delivery.

3. Formulations for Enteral Administration

Bioactive agents that are not peptides or polypeptides can also beformulated for oral delivery. Oral solid dosage forms are known to thoseskilled in the art. Solid dosage forms include tablets, capsules, pills,troches or lozenges, cachets, pellets, powders, or granules orincorporation of the material into particulate preparations of polymericcompounds such as polylactic acid, polyglycolic acid, etc. or intoliposomes. Such compositions may influence the physical state,stability, rate of in vivo release, and rate of in vivo clearance of thepresent proteins and derivatives. =Liposomal or polymeric encapsulationmay be used to formulate the compositions. In general, the formulationwill include the active agent and inert ingredients which protectpeptide in the stomach environment, and release of the biologicallyactive material in the intestine.

Another embodiment provides liquid dosage forms for oral administration,including pharmaceutically acceptable emulsions, solutions, suspensions,and syrups, which may contain other components including inert diluents;adjuvants such as wetting agents, emulsifying and suspending agents; andsweetening, flavoring, and perfuming agents.

Controlled release oral formulations may be desirable. Non-polypeptidebioactive agents can be incorporated into an inert matrix which permitsrelease by either diffusion or leaching mechanisms, e.g., films or gums.Slowly disintegrating matrices may also be incorporated into theformulation. Another form of a controlled release is one in which thedrug is enclosed in a semipermeable membrane which allows water to enterand push drug out through a single small opening due to osmotic effects.For oral formulations, the location of release may be the stomach, thesmall intestine (the duodenum, the jejunem, or the ileum), or the largeintestine. Preferably, the release will avoid the deleterious effects ofthe stomach environment, either by protection of the active agent (orderivative) or by release of the active agent beyond the stomachenvironment, such as in the intestine. To ensure full gastric resistancean enteric coating (i.e, impermeable to at least pH 5.0) is preferred.Examples of the more common inert ingredients that are used as entericcoatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films or as capsules such as thoseavailable from Banner Pharmacaps.

4. Drug Eluting Steals

The pharmaceutical composition containing a TGFβ inhibitor, a SMAD2inhibitor, an FGF Receptor agonist, a Let-7 agonist, or a combinationthereof, may also be released from a drug-eluting stent. A typicaldrug-eluting stent is a peripheral or coronary stent (a scaffold) placedinto narrowed, diseased peripheral or coronary arteries that slowlyreleases a drug to block cell proliferation. The disclosed stent cancontain a TGFβ inhibitor, a SMAD2 inhibitor, an FGF Receptor agonist, aLet-7 agonist, or a combination thereof instead of, or in addition to, adrug that blocks cell proliferation.

Drug-eluting stents have three parts: a stent platform, a coating, and adrug. The stent itself is generally an expandable metal alloy framework.Many drug-eluting stents are based on a bare-metal stent (BMS). Thestents have elaborate mesh-like designs to allow expansion, flexibilityand in some cases the ability to make/enlarge side openings for sidevessels. A coating, typically of a polymer, holds and elutes (releases)the drug into the arterial wall by contact transfer. Coatings aretypically spray coated or dip coated. There can be one to three or morelayers in the coating e.g. a base layer for adhesion, a main layer forholding the drug, and sometimes a top coat to slow down the release ofthe drug and extend its effect.

The main purpose of the drug is to inhibit neointimal growth which wouldcause restenosis. Existing drugs attempt to block proliferation ofsmooth muscle cells (SMC). The disclosed compositions containing a TGFβinhibitor, a SMAD2 inhibitor, an FGF Receptor agonist, a Let-7 agonist,or a combination thereof, to inhibit Endo-MT of endothelial cells intoSMC. Therefore, these compositions may also be used in combination withanti-proliferative drugs for a combinatorial effect. Immunosuppressiveand antiproliferative drugs may also be included.

5. Dosage

In one embodiment, the TGFβ TGFβ inhibitor, a SMAD2 inhibitor, an FGFReceptor agonist, a Let-7 agonist, or a combination thereof isadministered in a dose equivalent to parenteral administration of about0.1 ng to about 100 g per kg of body weight, about 10 ng to about 50 gper kg of body weight, about 100 ng to about 1 g per kg of body weight,from about 1 μg to about 100 mg per kg of body weight, from about 1 μgto about 50 mg per kg of body weight, from about 1 mg to about 500 mgper kg of body weight; and from about 1 mg to about 50 mg per kg of bodyweight. Alternatively, the amount of pharmaceutical compositionadministered to achieve a therapeutic effective dose is about 0.1 ng, 1ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg,17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90mg, 100 mg, 500 mg per kg of body weight or greater.

Area under the curve (AUC) refers to the area under the curve thattracks the serum concentration (nmol/L) of the TGFTGFβ inhibitor, aSMAD2 inhibitor, an FGF Receptor agonist, a Let-7 agonist, or acombination thereof over a given time following the IV administration ofthe reference standard. By “reference standard” is intended theformulation that serves as the basis for determination of the total doseto be administered to a human subject to achieve the desired positiveeffect, i.e., a positive therapeutic response that is improved withrespect to that observed without administration of the pharmaceuticalcomposition. In a preferred embodiment, the dose of the TGFβ inhibitor,Let-7 agonist, or combination thereof provides a final serum level ofTTGFβ inhibitor, a SMAD2 inhibitor, an FGF Receptor agonist, a Let-7agonist, or a combination thereof of about 100 ng/ml to about 1000ng/ml, about 1100 ng/ml to about 1450 ng/ml, 100 ng/ml to about 250ng/ml, about 200 ng/ml to about 350 ng/ml, about 300 ng/ml to about 450ng/ml, about 350 ng/ml to about 450 ng/ml, about 400 ng/ml to about 550ng/ml, about 500 ng/ml to about 650 ng/ml, about 600 ng/ml to about 750ng/ml, about 700 ng/ml to about 850 ng/ml, about 800 ng/ml to about 950ng/ml, about 900 ng/ml to about 1050 ng/ml, about 1000 ng/ml to about1150 ng/ml, about 100 ng/ml to about 1250 ng/ml, about 1200 ng/ml toabout 1350 ng/ml, about 1300 ng/ml to about 1500 ng/m. In specificembodiments, the serum level of the TGFβ inhibitor, a SMAD2 inhibitor,an FGF Receptor agonist, a Let-7 agonist, or a combination thereof isabout 100 ng/ml, 250 ng/ml, 300 ng/ml, 350 ng/ml, 360 ng/ml, 370 ng/ml,380 ng/ml, 390 ng/ml, 400 ng/ml, 410 ng/ml, 420 ng/ml, 430 ng/ml, 440ng/ml, 450 ng/ml, 500 ng/ml, 750 ng/ml, 900 ng/ml, 1200 ng/ml, 1400ng/ml, or 1600 ng/ml.

Pharmaceutical formulations can be designed for immediate release,sustained release, delayed release and/or burst release of one or moreactive agents in a therapeutically effective amount. In a preferredembodiment, the formulation provides an initial burst release of a“loading dosage”, followed by a sustained release to maintain thetherapeutically effective dosage. This can be accomplished using adelayed and/or extended release formulation.

III. Methods for Inhibiting Neointima Stenosis

Methods are disclosed for inhibiting neointima stenosis. In someembodiments, the neointima stenosis results from injury to the vascularwall (endothelium) after a medical procedure, such as angioplasty,vascular graft implantation, or stent placement. The method involvesinhibiting the endothelial to mesenchymal transition (Endo-MT) ofvascular endothelial cells into smooth muscle cells (SMC). FGF signalingsuppresses Endo-MT by maintaining Let-7 microRNA expression, whichprevents the activation of TGFβ, TGFβR1 and SMAD2 expression. Therefore,the disclosed method involves the use of a composition containing a TGFβinhibitor, a SMAD2 inhibitor, an FGF Receptor agonist, a Let-7 agonist,or a combination thereof to inhibit Endo-MT at the site of actual orpotential neointima formation.

In some embodiments, the composition is coated on or incorporated into avascular device, such as a vascular graft, angioplasty balloon, orvascular stent prior to its use. The composition may coated on orincorporated into the vascular device using any known suitable method.The composition may be encapsulated in the form of microspheres,nanospheres, microparticles and/or microcapsules, and seeded on or intothe vascular device.

In other embodiments, the composition is administered directly to thesubject before, during, or after a medical procedure that may damage avascular wall. In these embodiments, the composition is preferablyadministered locally to the site of actual or potential neointimaformation.

A. Administration

In some embodiments, a pharmaceutical composition containing a TGFβinhibitor, a SMAD2 inhibitor, an FGF Receptor agonist, a Let-7 agonist,or a combination thereof is administered to a subject receiving avascular device in an effective amount to prevent, inhibit or reduceneointimal stenosis. The precise dosage will vary according to a varietyof factors such as the nature of the compound being administered, theroute of administration, and subject-dependent variables (e.g., age,etc.).

The pharmaceutical composition may be administered systemically orlocally at the site of vascular graft implantation. The compositions maybe administered prior to vascular device implantation, at the same timeas vascular device implantation, following vascular device implantation,or any combination thereof. In one embodiment, the composition isadministered either locally or systemically from a controlled releaseformulation. The composition may be administered separately fromadditional bioactive agents or may be co-administered.

Pharmaceutical compositions containing a TGFβ inhibitor, a SMAD2inhibitor, an FGF Receptor agonist, a Let-7 agonist, or a combinationthereof may be administered by parenteral (intramuscular,intraperitoneal, intravenous (IV) or subcutaneous injection),transdermal (either passively or using iontophoresis orelectroporation), or transmucosal (nasal, vaginal, rectal, orsublingual) routes of administration. The compositions can be formulatedin dosage forms appropriate for each route of administration.Compositions containing bioactive agents that are not peptides orpolypeptides can additionally be formulated for enteral administration.

IV. Methods for Promoting Patency of Biodegradable, Synthetic VascularGrafts

The patency of biodegradable, synthetic vascular grafts may be increasedusing a composition that inhibits Endo-MT. Therefore, a method forpromoting patency of a biodegradable, synthetic vascular graft isdisclosed that involves administering a composition that inhibitsEndo-MT to the graft or to the subject prior to or after implantation.Also disclosed is a cell-free tissue engineered vascular graft (TEVG)produced by this method.

Suitable Endo-MT inhibitors include TGFTGFβ inhibitor, a SMAD2inhibitor, an FGF Receptor agonist, a Let-7 agonist, or a combinationthereof. The administration of Endo-MT inhibitors increases the patencyof the biodegradable, synthetic vascular grafts.

The disclosed biodegradable, synthetic vascular grafts do not requirecell seeding to maintain patency of the grafts. This is advantageous,because it avoids problems associated with cell seeding, including theneed for an invasive procedure to obtain autologous cells, the need fora substantial period of time in order to expand the cells in culture,the inherent difficulty in obtaining healthy autologous cells fromdiseased donors, and an increased risk of contamination and thepotential for dedifferentiation of the cells. The disclosedbiodegradable, synthetic vascular grafts therefore have a greater theoff-the-shelf availability and increased overall clinical utility.

A. Polymeric Vascular Grafts

The disclosed polymeric vascular grafts are typically tubular porousconduits fabricated using biodegradable polymers. The pores in thepolymeric vascular grafts allow for recruitment and integration of hostcells into the graft. It is believed that recruited host cells mediateoutward vascular tissue remodeling and vascular neotissue formation.Unlike synthetic vascular grafts that are currently in clinical use, thedisclosed polymeric vascular grafts are biodegradable, which allows forthe grafts to become replaced by forming neotissue as they degrade.Thus, the disclosed polymeric vascular grafts offer growth potentialthat is not possible with currently used synthetic vascular grafts.

The disclosed grafts are preferably substantially tubular in shape witha round or substantially round cross section. The tubular grafts have alumen extending throughout the length of the graft. The grafts may be ofany appropriate length and diameter that is suitable for the intendedsurgical use of the graft. Typically, the graft should be slightlylonger than the length of artery or vein that is to be replaced.

The porous polymeric vascular grafts may be fabricated using anyappropriate method, such as melt processing, solvent processing,leaching, foaming, extrusion, injection molding, compression molding,blow molding, spray drying, extrusion coating, and spinning of fiberswith subsequent processing into woven or non-woven constructs. Pores inthe graft may be derived by any suitable method, including saltleaching, sublimation, solvent evaporation, spray drying, foaming,processing of the materials into fibers and subsequent processing intowoven or non-woven devices. Preferably, the pores of the device arebetween 5 μm and 500 μm, more preferably between 5 μm and 250 μm, morepreferably between 5 μm and 100 μm, in diameter.

In some embodiments, the grafts are formed from a felt or sheet likematerial of the polymer that can be formed into a tubular conduit. Forexample the device could be fabricated as a nonwoven, woven or knittedstructure from extruded polymeric fibers. The polymeric sheet may beformed using any textile construction, including, but not limited to,weaves, knits, braids or filament windings. Any suitable method, such aselectrospinning, may be used to fabricate the nonwoven or wovenpolymeric textile.

The polymers and fabrication methods selected to fabricate the polymericvascular grafts are suitable to produce grafts with biomechanicalproperties suitable for use as vascular conduits. Biomechanicalproperties that are important for vascular graft function includeinitial burst pressure, suture retention strength and elasticity. Insome embodiments, the initial burst pressure of the polymeric vasculargraft is between about 1,500 mmHg and about 4,500 mmHg, preferablybetween about 2,000 mmHg and about 4,500 mmHg. In another embodiment,the polymeric vascular grafts possess suture retention strengths betweenabout 1 N and about 5 N, preferably between about 2 N and about 4 N. Inanother embodiment, the intrinsic elasticity of the vascular grafts isbetween about 10 MPa and about 50 MPa, preferably between about 15 MPaand about 40 MPa. In another embodiment, the initial tensile strength ofthe vascular grafts is between about 1 MPa and about 10 MPa, preferablybetween about 3 MPa and about 6 MPa.

1. Biodegradable Polymers

The biodegradable, synthetic vascular grafts may be fabricated using anyknown biodegradable polymer, co-polymer, or mixture thereof. Manysuitable biodegradable polymers are known in art.

Examples of preferred biodegradable polymers include synthetic polymersthat degrade by hydrolysis such as poly(hydroxy acids), such as polymersand copolymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyesters, polyurethanes, poly(butic acid),poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), andpoly(lactide-co-caprolactone). The foregoing materials may be usedalone, as physical mixtures (blends), or as co-polymers. In a preferredembodiment, the biodegradable, synthetic vascular grafts are fabricatedfrom polyglycolic acid or poly-L-lactic acid.

2. Sealants

Synthetic vascular grafts fabricated from nonwoven, woven or knittedsheets or felts of biodegradable polymers may be further treated withpolymeric sealants. The polymeric sealants function to modify thebiomechanical properties of the vascular grafts, such as tensilestrength and elasticity. Polymeric sealants may also be used to controlthe total porosity and pore size distribution range of the vasculargraft.

Polymeric sealants for the disclosed biodegradable synthetic vasculargrafts may be any biodegradable polymer, including, but not limited to,the list of biodegradable polymers listed above. In one embodiment, thepolymeric sealant is a copolymer of poly(ε-caprolactone) andpoly(L-lactide). The copolymer can be at a ratio from 5:95 to 95:5,preferably from 30:70 to 70:30, more preferably from 40:60 to 60:40,most preferably about 50:50.

Polymeric sealants may be added to tubular synthetic grafts dissolved inan appropriate solvent to allow for the sealant to penetrate thenonwoven, woven or knitted sheet or felt of biodegradable polymersforming the graft. The polymeric sealant may then be quickly transformedfrom liquid to solid phase by lowering the temperature of the graft.Solvents may then be removed by an appropriate technique, such aslyophilization.

C. Additional Bioactive Agents

Additional bioactive agents that promote vascular graft adaptation mayalso be administered. Suitable bioactive agents or drugs include, butare not limited to: anti-thrombogenic agents, such as heparin, heparinderivatives, urokinase, and PPack (dextrophenylalanine praline argininechloromethylketone; anti-proliferative agents, such as enoxaprin,angiopeptin, hirudin, acetylsalicylic acid, paclitaxel, 5-fluorouracil,cisplatin, vinblastine, vincristine, epothilones, endostatin,angiostatin and thymidine kinase inhibitors; anesthetic agents, such aslidocaine, bupivacaine, and ropivacaine; anti-coagulants, such asD-Phe-Pro-Arg chloromethyl keton, RGD peptide-containing compounds,heparin, antithrombin compounds, platelet receptor antagonists,anti-thrombin antibodies, anti-platelet receptor antibodies, aspirin,prostaglandin inhibitors, platelet inhibitors and tick antiplateletpeptides; cholesterol-lowering agents; vasodilating agents; and agentswhich interfere with endogenous vascoactive mechanisms.

D. Uses for Biodegradable, Synthetic Vascular Grafts

The disclosed biodegradable, synthetic vascular grafts may be used asvenous, arterial or artero-venous conduits for any vascular orcardiovascular surgical application. Exemplary applications include, butare not limited to, congenital heart surgery, coronary artery bypasssurgery, peripheral vascular surgery and angioaccess.

Vascular bypass grafting is most commonly performed for the treatment ofvessel stenosis. However, vascular grafts are also used for thetreatment of other conditions such as arterial aneurysm or chronic renalfailure (as access for hemodialysis). Vascular grafting can be performedby conventional surgery or endovascular techniques.

Coronary artery bypass grafting (CABG) is one example of vascular bypasssurgery. With this procedure, a bypass graft is used to bypass thecoronary artery distal to the site of stenosis or occlusion. When a veingraft is used, one end is anastomosed to the aorta and the other end isanastomosed to the coronary artery beyond the stenosis or occlusion.When an arterial graft is used, the proximal end is left undisturbed(thus preserving the artery's normal blood inflow) and the distal end isanastomosed to the coronary artery beyond the stenosis or occlusion.

Typically, an anastomosis (i.e., the surgical union of tubular parts)between the in situ artery or vein and the synthetic graft is created bysewing the graft to the in situ vessel with suture. Commonly used suturematerials include proline (extruded polypropyline) and ePTFE.

EXAMPLES Example 1: Basal FGF Signaling Suppresses TGFβ-Mediated Endo-MT

Materials and Methods

Cell Culture and Reagents

Human 293T T17 cells (ATCC CRL-11268) were maintained in Dulbecco'smodified Eagle's medium (Gibco) with 10% fetal bovine serum (Invitrogen)and penicillinstreptomycin (Gibco). Primary human mammary epithelialcells (HuMEC, Invitrogen), were maintained in HuMEC basal serum freemedium with HuMEC supplement (Invitrogen 12754-016) and bovine pituitaryextract (Invitrogen 13028-014). Human foreskin cells (ATCC CRL-1079Sk)were maintained in Minimum Essential Medium (ATCC) with 10% fetal bovineserum (Hyclone), β-mercaptoethanol (Sigma), and penicillin-streptomycin(Gibco). Primary human atrial cells (passage 3-5; gift from Y. Qyang,Yale University School of Medicine) were maintained in Dulbecco'smodified Eagle's medium (Gibco) with 10% fetal bovine serum(Invitrogen), L-glutamine (Gibco), Sodium pyruvate (Gibco),Non-essential amino acid (Gibco), and penicillin-streptomycin (Gibco).HUAEC (human umbilical artery endothelial cells, passage 5-10, Lonza)were cultured in EBM-2 supplemented with EGM-2-MV bullet kit (Lonza).HUVEC (human umbilical vein endothelial cells, passage 3-7, YaleVascular Biology and Therapeutics) were cultured in M199 with ECGS,supplemented with 20% FBS (Sigma), L-glutamine (Gibco), andpenicillin-streptomycin (Gibco). Culture vessels were coated with 0.1%gelatin (Sigma) for 30 min at 37° C. immediately prior to cell seeding.Primary mouse endothelial cells were isolated from the heart using ratanti-mouse CD31 antibody and Dynabeads (Invitrogen) as previouslydescribed (Partovian C, et al. Mol Cell. 32(1):140 (2008)).

Antibodies Used for Immunodetection of Proteins

The following antibodies were used for flow cytometry (F),immunoblotting (IB), immunofluorescence (IF), or immunohistochemistry(IHC): antibodies to 5G11 (IB; Mood K, et al. J Biol Chem. 277(36):33196(2002)), β-galactosidase (Ab9361, Abeam; IF), Calponin, clone hCP(C-2687, Sigma; IB), CD31 (sc-1506, Santa Cruz; IB), CD31 (Ab28364-100,Abeam; IF), CD31 (550274, BD Pharmingen; EC isolation), CD31-APC(FAB3567A, R&D; F), FLAG (F1804, Sigma; IB), FRS2 (H-91, Santa Cruz;IB), Lin28A (3695, Cell Signaling; IB), N-cadherin (610920, BDTransduction Laboratories; IB), Smad2 (3122, Cell Signaling; IB),p-Smad2 (Ser465/467) (3108, Cell Signaling; IB), SM22 alpha (ab14106,Abeam; IB), smooth muscle α-actin clone 1A4 (A2547, Sigma; IB), smoothmuscle α-actin (M0851, Dako; IF), TGFβ (Ab53169, Abeam; IHC), TGFβR1(sc-398, Santa Cruz; IB), β-tubulin (T7816, Sigma; IB), VE-cadherin(C-19, Santa Cruz; IB, IF), Vimentin clone V9 (V6630, Sigma; IB), VEGFR2(2479, Cell Signaling; IB), ZEB1 (HPA027525, Sigma; IB).

Plasmid and Lentivirus Constructs

TGFβR1, TGFβR1 T202D, and TGFβR1 K230R were purchased from Addgene andsubcloned into the lentivirus pLVX-IRES-Puro vector (Clontech).BamH1-cleaved cDNAs fragments encoding Xenopus dominant negative FGFR1,constitutively active FGFR1 K562E, and its derivative mutants wereinserted into the lentivirus pLVX-IRESPuro vector. Plasmids containingmultiple mutations were generated using the QuickChange site-directedmutagenesis kit (Stratagene) employing the mouse FRS2 construct astemplate according to the manufacturer's recommendation. The FRS2mutants were introduced using the primers in Table 1.

TABLE 1 Primers for introducing FRS1 mutants PrimerPrimer Sequence (5′→3′) FRS2 GCTGAGGAAC AAGTACATAC SEQ ID Y196FTTTTGTTAAC ACTACAGGTG NO: 1 TGCAA CTTGCACACC TGTAGTGTTA SEQ IDACAAAAGTAT GTACTTGTTC NO: 2 CTCAGC FRS2 GCCCCCTGTC AACAAACTGG SEQ IDY306F TGTTTGAAAA TATAAACGGG NO: 3 CTATCTATTC GAATAGATAG CCCGTTTATASEQ ID TTTTCAAACA CCAGTTTGTT NO: 4 GACAGGGGGC FRS2 GAGAAGACCT GCACTATTAASEQ ID Y349F ACTTTGAAAA TTTACCATCT NO: 5 TTGCCTCC GGAGGCAAAG ATGGTAAATTSEQ ID TTCAAAGTTT AATAGTGCAG NO: 6 GTCTTCTC FRS2 GATCCAATGC ATAACTTTGTSEQ ID Y392F TAATACAGAG AATGTAACAG NO: 7 TGCCG CGGCACTGTT ACATTCTCTGSEQ ID TATTAACAAA GTTATGCATT NO: 8 GGATC FRS2 GTTTAGAACA TAGGCAACTCSEQ ID Y436F AATTTTATAC AGGTGGATTT NO: 9 GGAAGG CCTTCCAAAT CCACCTGTATSEQ ID AAAATTGAGT TGCCTATGTT NO: 10 CTAAAC FRS2 CACGCGGCGC ACAGAGCTGTSEQ ID Y471F TCGCTGTGAT AGACATTGAG NO: 11 AGAA TTCTCTCAAT GTCTATCACASEQ ID GCGAACAGCT CTGTGCGCCG NO: 12 CGTGThe constructs were verified by sequence analysis (Yale Keck DNAsequencing core facility), and protein expression was confirmed byimmunoblot analysis.

Generation of Lentiviruses

The production of FRS2 lentivirus was previously described (Murakami JCIin press). For the production of miRNA lentivirus, 10 μg of pMIRNA1carrying the let-7 miRNA expression cassette (System Biosciences), 5 μgof pMDLg/PRRE, 2.5 μg of RSV-REV, and 3 μg of pMD.2G were co-transfectedinto 293T cells using Fugene 6. Forty eight hr later, the medium washarvested, cleared by 0.45 μm filter, mixed with polybrene (Sigma), andapplied to the cells. After 6 hr incubation, the virus-containing mediumwas replaced by fresh medium.

Western Blot Analysis

Tissues and cells were lysed with T-PER (Thermo scientific) containingcomplete mini protease inhibitors (Roche) and phosphatase inhibitors(Roche). 20 μg of total protein from each sample was resolved on a4%-12% Bis-Tris Gel (Bio-Rad) with MOPs Running Buffer (Bio-Rad) andtransferred to nitrocellulose membranes (Bio-Rad). The blots were thenprobed with various antibodies. Chemiluminescence measurements wereperformed using SuperSignal West Femto Maximum Sensitivity Substrate(Thermo Fisher Scientific).

RNA Isolation, qRT-PCR, and Gene Expression Profiling

RNA was isolated using RNeasy® plus Mini Kit (Qiagen) and converted tocDNA using iScript™ cDNA synthesis kit (Bio-Rad). Quantitative real-timePCR (qRT-PCR) was performed using Bio-Rad CFX94 (Bio-Rad) by mixingequal amount of eDNAs, iQ™ SYBR® Green Supermix (Bio-Rad) and genespecific primers. All reactions were done in a 25 μl reaction volume induplicate. Data were normalized to endogenous β-actin. Values areexpressed as fold change in comparison to control. Primers are listed inTable 2. Mouse TGFβ gene expression was quantified via qRT-PCR withTaqMan® Gene Expression Assays (Mm00441726_m1; Applied Biosystems) andHPRT (Mm00441258_1; Applied Biosystems) as endogenous control followedthe manufacturer's recommendation. Quantitative PCR analysis of 84 TGFβand epithelial to mesenchymal transition (EMT) related genes wereperformed using Human TGFβ BMP signaling pathway (PAHS-035D, QIAGEN) andHuman Epithelial to Mesenchymal Transition (PAHS-090D, QIAGEN) RT²Profiler™ PCR Arrays following the manufacturer's protocol. RT²Profiler™ PCR arrays were run on a Bio-Rad CFX96 machine (Bio-Rad).Duplicate arrays were run per condition for control and FRS2 knockdownhuman umbilical arterial endothelial cells (HUAEC). Data analysis wasperformed using the manufacturer's integrated web-based software packagefor the PCR Array System using cycle threshold (Ct)-based fold-changecalculations.

TABLE 2 Primers used for quantitative RT-PCRanalysis of mouse and human genes Gene Primer sequence 5′→3′ MouseGGCATCTGTAAGTGGTTCAACG SEQ ID Lin28 NO: 13 CCCTCCTTGAGGCTTCGGA SEQ IDNO: 14 Mouse GATGGGCTCTCTCCAGATCAG SEQ ID Myocardin NO: 15GGCTGCATCATTCTTGTCACTT SEQ ID NO: 16 Mouse GGGCTGTGCTGTCTGTTGA SEQ IDNG2 NO: 17 TGATTCCCTTCAGGTAAGGCA SEQ ID NO: 18 MouseTCCATGCTAGACTCAGAAGTCA SEQ ID PDGFRα NO: 19 TCCCGGTGGACACAATTTTTC SEQ IDNO: 20 Mouse TTCCAGGAGTGATACCAGCTT SEQ ID PDGFRβ NO: 21AGGGGGCGTGATGACTAGG SEQ ID NO: 22 Mouse GCAGGCAACGATGAAAACTACT SEQ IDRunx1 NO: 23 GCAACTTGTGGCGGATTTGTA SEQ ID NO: 24 MouseGTCCCAGACATCAGGGAGTAA SEQ ID SM α- NO: 25 actin TCGGATACTTCAGCGTCAGGASEQ ID NO: 26 Mouse CAACAAGGGTCCATCCTACGG SEQ ID SM22_ NO: 27ATCTGGGCGGCCTACATCA SEQ ID NO: 28 Mouse AAACAAGAGCGGAGATTTGAGC SEQ IDSM- NO: 29 calponin TGTCGCAGTGTTCCATGCC SEQ ID NO: 30 MouseAAGCTGCGGCTAGAGGTCA SEQ ID SM- NO: 31 MHC CCCTCCCTTTGATGGCTGAG SEQ IDNO: 32 Mouse TCCCAACTACAGGACCTTTTTCA SEQ ID TGFβRI NO: 33GCAGTGGTAAACCTGATCCAGA SEQ ID NO: 34 Mouse CGGCTGCGAGAGAAATTGC SEQ IDVimentin NO: 35 CCACTTTCCGTTCAAGGTCAAG SEQ ID NO: 36 MouseGTTGTCGACGACGAGCG SEQ ID β-actin NO: 37 GCACAGAGCCTCGCCTT SEQ ID NO: 38Human AACAGTGTTGACATGAAGAGCC SEQ ID CD31 NO: 39 TGTAAAACAGCACGTCATCCTTSEQ ID NO: 40 Human TTCTGTTCGCAGGTGATTGG SEQ ID Collagen NO: 41 1A1CATGTTCAGCTTTGTGGACC SEQ ID NO: 42 Human AACTTGTCACCCTCTTTGCC SEQ IDFSP1 NO: 43 TCCTCAGCGCTTCTTCTTTC SEQ ID NO: 44 HumanAAACCAATTCTTGGAGCAGG SEQ ID Fibro- NO: 45 nectin CCATAAAGGGCAACCAAGAGSEQ ID NO: 46 Human GATCCAACTGCTGCTGAGGT SEQ ID HMGA2 NO: 47AGGCAGACCTAGGAAATGGC SEQ ID NO: 48 Human CGGGCATCTGTAAGTGGTTC SEQ IDLin28 NO: 49 CAGACCCTTGGCTGACTTCT SEQ ID NO: 50 Human N-GAGGAGTCAGTGAAGGAGTCA SEQ ID cadherin NO: 51 GGCAAGTTGATTGGAGGGATGSEQ ID NO: 52 Human GCCATCACCACTCAAAACTGTG SEQ ID Smad2 NO: 53CCTGTTGTATCCCACTGATCTA SEQ ID NO: 54 Human SM CAAAGCCGGCCTTACAGAG SEQ IDα-actin NO: 55 AGCCCAGCCAAGCACTG SEQ ID NO: 56 HumanGATTTTGGACTGCACTTCGC SEQ ID SM22α NO: 57 GTCCGAACCCAGACACAAGT SEQ IDNO: 58 Human SM- CTGGCTGCAGCTTATTGATG SEQ ID Calponin NO: 59CTGAGAGAGTGGATCGAGGG SEQ ID NO: 60 Human CAAGCAGAGTACACACAGCAT SEQ IDTGFβ1 NO: 61 TGCTCCACTTTTAACTTGAGCC SEQ ID NO: 62 HumanACGGCGTTACAGTGTTTCTG SEQ ID TGFβRI NO: 63 GCACATACAAACGGCCTATCT SEQ IDNO: 64 Human TCTGGTTGTCACAGGTGGAA SEQ ID TGFβRII NO: 65GCACGTTCAGAAGTCGGTTA SEQ ID NO: 66 human VE- CATGAGCCTCTGCATCTTCC SEQ IDcadherin NO: 67 ACAGAGCTCCACTCACGCTC SEQ ID NO: 68 HumanCGGCTCTTTCGCTTACTGTT SEQ ID VEGFR2 NO: 69 TCTCTCTGCCTACCTCACCTG SEQ IDNO: 70 Human GCAAAGATTCCACTTTGCGT SEQ ID Vimentin NO: 71GAAATTGCAGGAGGAGATGC SEQ ID NO: 72 Human CAGTCAGCTGCATCTGTAACAC SEQ IDZEB1 NO: 73 CCAGGTGTAAGCGCAGAAAG SEQ ID NO: 74 Human CAAGAGGCGCAAACAAGCCSEQ ID ZEB2 NO: 75 GGTTGGCAATACCGTCATCC SEQ ID NO: 76 Human β-ATCAAGATCATTGCTCCTCCTGA SEQ ID actin NO: 77 GCTGCTTGCTGATCCACATCTGSEQ ID NO: 78

MicroRNA Target Prediction

To identify potential miRNA binding sites within the 3′UTR of TGFβR1,the following bioinformatic databases were used: TargetScan, PitTar, andDIANA-microT v3.0.

MicroRNA Real-Time PCR Analysis

Quantitative PCR analysis of 88 miRNA was performed using Human CellDifferentiation & Development RT² Profiler™ PCR Arrays (MAH-103A,QIAGEN) following the manufacturer's protocol. Validation of microRNAarray data was performed with the RT² miRNA qPCR Assays and RT-PCRPrimer Sets according to the manufacturer's instructions (QIAGEN).Primers for human mature let-7a (MPH00001A), let-7b (MPH00002A), let-7c(MPH00003A), let-7d (MPH00004A), let-7e (MPH00005A), let-7f (MPH00006A),let-7g (MPH00007A), mir-98 (MPH00480A), SNORD47 (MPH01660A) were fromQIAGEN. Primers for mouse mature let-7a (MPM00483A), let-7b (MPM00484A),let-7c (MPM485A), let-7d (MPM00486A), let-7e (MPM00487A), let-7f(MPM00488A), let-7g (MPM00489A), let-7i (MPM00490A), mir-98 (MPM00875A)were from QIAGEN. Individual miRNA expression was normalized in relationto expression or small nuclear U6B RNA, a commonly used internal controlfor miRNA quantification assay. PCR amplification consisted of 10 min ofan initial denaturation step at 95° C., followed by 45 cycles of PCR at95° C. for 30 s, 60° C. for 30 s.

Flow Cytometry

Cells were incubated with 10 μg/ml Dil-Ac-LDL for 4 hr at 37° C. Cellswere then trypsinized, stained with APC-conjugated anti-PECAM-1 antibodyor isotype control (R&D) and analyzed by FACScan™ (Becton Dickinson) andFlow-Jo Software (TreeStar). Two independent experiments were performedby duplicate.

Transfection of miRNA Mimics and Luciferase Reporter Assays

For reporter assays, cells were plated in 6-well plates andco-transfected with 1 μg let-7 core 4 firefly luciferase reporterplasmid using Fugene® 6 (Roche). After 24 hr, cells were transfectedwith let-7 miRNA mimic (10 nM; QIAGEN) or control oligonucleotides (10nM; QIAGEN) using HiPerFect® reagent (QIAGEN). After 24 hr ofincubation, cells were lysed in passive lysis buffer (Promega) andluciferase activity was measured with the Dual-Luciferase Reporter AssaySystem (Promega) using TD-20/20 Luminometer (Turner Designs).

Growth Factors and Chemicals

Recombinant human FGF2 (R&D) and TGFβ1 (Peprotech) were reconstituted in0.1% BSA/PBS. U0126 (Sigma), a MEK inhibitor, was reconstituted in DMSOand used at a final concentration of 10 μM. SB431542 (Sigma), a TGFBR1kinase inhibitor, was reconstituted in DMSO and used at a finalconcentration of 10 μM in cell culture. Actinomycin D (Sigma), a DNAtranscription suppressor, was reconstituted in acetone and used at afinal concentration of 10 μg/ml in cell culture. Cycloheximide (Sigma),a protein synthesis inhibitor, was reconstituted in DMSO and used at afinal concentration of 10 μM in cell culture.

Generation of Mice and Embryos

FRS2^(flox/flox) (Lin Y, et al. Genesis. 45(9):554 (2007)) and FRS2α 2F(Gotoh N, et al. Proc Natl Acad Sci USA. 101(49):17144 (2004)) mice werepreviously described. Tie2-Cre transgenic mice or Cdh5-CreERT2transgenic mice were crossed with R26R-lacZ[B6,129-Gt(ROSA)26Sor^(tm1Sho/J)] (JAX SN:003309) or mT/Mg[B6,129(Cg)-Gt(ROSA)26Sor^(tm4(ACTB-tdTomato,-EGFP)Luo)/J] JAXSN:007676) mice to generate endothelial cell-specific reporter mice. Forembryo analysis, timed matings were set up and the morning of thevaginal plug was considered as embryonic day 0.5 (E0.5). Embryos weregenotyped by PCR analysis of the yolk sacs. PCR genotyping was performedusing the primers in Table 3.

TABLE 3 Primers for PCT geno- typing FRS2^(flox/flox) mice PrimerPrimer Sequence (5′→3′) FRS2 2F AGAATGGTGGCAC SEQ ID AAACCAATAATCCNO: 79 CAATTCTTAACAC SEQ ID CCACAAGGCCG NO: 80 FRS2^(flox/flox)GAGTGTGCTGTGA SEQ ID TTGGAAGGCAG NO: 81 GCACGAGTGTCTG SEQ ID CAGACACATGNO: 82 Rosa26 GCGAAGAGTTTGT SEQ ID CCTCAACC NO: 83 AAAGTCGCTCTGA SEQ IDGTTGTTAT NO: 84 GGAGCGGGAGAAA SEQ ID TGGATATG NO: 85 Tie2-CreGCGGTCTGGCAGT SEQ ID AAAAACTATC NO: 86 GTGAAACAGCATT SEQ ID GCTGTCACTTNO: 87 CTAGGCCACAGAA SEQ ID TTGAAAGATCT NO: 88 GTAGGTGGAAATT SEQ IDCTAGCATCATCC NO: 89

Statistical Analysis

Statistical comparisons between groups were performed by the one-wayanalysis of variance followed by the Student t-test. P values less than0.05 were considered significant.

Results

To test the role of FGF signaling in EC, RNA interference was used toinhibit the expression of FRS2. Immunofluorescence staining showed thatwhile control HUAEC display a typical rounded/cobblestone morphology,after FRS2 knockdown there was a distinct change in cell shapeaccompanied by expression of smooth muscle calponin (SM-calponin), aprotein not normally expressed in the endothelium, while an endothelialmarker VE-cadherin was still expressed. Western blotting confirmed theappearance of this and other smooth muscle cell markers in FRS2knockdown cells. FACS analysis showed that both control and FRS2knockdown EC expressed CD31 and were able to take up Dil-acLDL, rulingout smooth muscle cell contamination. Similar results were obtained inprimary mouse FRS2^(flox/flox) EC following Ad-Cre transduction (FIGS.9A-9J).

These finding indicated that following FRS2 knockdown EC are undergoingEndo-MT. Since TGFβ signaling has been implicated in Endo-MT, theexpression of genes involved in TGFβ signaling was examined. qPCRanalysis demonstrated a marked increase in expression of TGFβ1 andTGFβ2, all three TGFβ receptors and several collagen isoforms that arestimulated by TGFβ signaling (Table 4; FIGS. 1A-1F). Western blottingconfirmed increased expression and activation of TGFβ signaling.Transduction of HUAEC with dominant negative FGFR1 or FRS2 mutantsresulted in a similar increase in TGFβ signaling and smoothmuscle/mesenchymal marker expression (FIGS. 10A-10F).

TABLE 4 mRNA expression profiles in HUAECs after FRS2 knockdown Genes(Fold Change) Up-Regulated FRS2 shRNA/Controls BMP4 5.26 TGFβ1 6.3 TGFβ253.3 IGF1 147.65 IGFBP3 244.89 COL1A1 28.56 COL1A2 5.01 COL3A1 6.76

To confirm the relevance of our findings beyond the primary EC, FRS2 wasknocked down in primary human mammary epithelial cells (HuMEC) that aretypically used in EMT studies. mRNA expression profiles were assessedusing quantitative PCR arrays with cDNA from control or FRS2 knockdowncells. Genes with a statistically significant up-regulation greater than5-fold are shown in Table 5. Similar to observations in EC, FRS2knockdown in HuMEC resulted in the change from the normal roundedepithelial cell to a spindle-like mesenchymal phenotype, increasedexpression of mesenchymal markers including vimentin, N-cadherin, snail,ZEB1 (zinc finger E-box binding homebox 1) and ZEB2, and decreasedexpression of EMT repressor miRNAs mir-200b and mir-200c (FIGS.11A-11M). FRS2 knockdown in human foreskin and atrial cells alsoresulted in increased TGFβ signaling (FIGS. 12A-12H). Collectively,these data support the hypothesis that basal FGF signaling inhibitsTGFβ-mediated Endo-MT.

TABLE 5 mRNA expression profiles in HuMECs after FRS2 knockdown GenesUp-Regulated (Fold Change) During EMT FRS2 shRNA/Control CDH2 5.4 MMP322.82 SNAI1 12.4 ZEB1 14.95 ZEB2 14.05

To demonstrate that activation of TGFβ signaling following FRS2knockdown is indeed necessary for Endo-MT, three different experimentalapproaches were used to silence it. Treatment of FRS2 knockdown EC withTGFβR1 inhibitor SB431542 significantly decreased SM-calponin,fibronectin and vimentin expression (FIGS. 2A-2C). Similar results wereseen after transduction of a dominant negative TGFβR1 construct (TGFβR1K230R) (FIGS. 2D-2F). Finally, since SMAD2 activation was detectedfollowing FRS2 knockdown, its expression was silencing using a specificshRNA. This resulted in a significant decrease in SM-calponin, but notin fibronectin and vimentin expression (FIGS. 2G-2I).

Example 2: FGF Signaling Regulates TGFβR1 mRNA Stability Via Regulationof Let7 miRNA Expression

Next, the mechanism responsible for increased TGFβR1 and SMAD2expression following FGF signaling shutdown was examined. Quantitativeanalyses of TGFβR1 and Smad2 mRNA half-life showed a marked increasefollowing a knockdown of FRS2 expression (FIGS. 3A-3B). This indicatesthe presence of miRNA regulating the stability of these messages. Insilico analysis of TGFβR1 and Smad2 messages identified binding sitesfor let-7 family of miRNAs (SEQ ID NO:91) with an exact match to theseed sequence of let-7 in TGFβR1 3′-UTR 75-82 (SEQ ID NO:99) and3889-3895 (SEQ ID NO:100) and in Smad2 3′-UTR 3771-3777 (SEQ ID NO:101)downstream from the stop codon. miRNA array analysis demonstrated a 24to 120 fold reduction in expression of all let-7 family membersfollowing FRS2 knockdown that was confirmed by qPCR (FIGS. 3C-3H, Table6).

TABLE 6 mRNA expression of let-7 after FRS2 knockdown (Fold Change)let-7 family FRS2 shRNA/Control hsa-let-7a −26.26 hsa-let-7b −108.01hsa-let-7c −119.84 hsa-let-7d −37.66 hsa-let-7e −41.5 hsa-let-7f −23.83hsa-let-7g −24.34 hsa-let-7i −66.49

To confirm that FOP signaling controls let-7 miRNA expression, mouseembryos with defective FGF signaling were examined following a knock-inof a mutant FRS2a construct (FRS2a 2F/2F). A gene dosage-dependentreduction in let-7 levels was observed (FIGS. 3H-3N).

Example 3: Let-7 Regulates TGFβR1 Expression in Endothelial Cells

Materials and Methods

In Vivo Let-7 Antagomir Delivery

Mice were administrated with either phosphate buffered saline (PBS) orAF12 complexes at 2 mg/kg body weight in 0.2 ml per injection vialateral tail vein. Measurements of miRNA or mRNA levels in endothelialcells were performed 6 days after the injection.

Results

To further explore let-7 role in regulation of TGFβ signaling, theexpression of let-7 family members was inhibited in an FGF-independentmanner. Transfection of Lin28 (FIG. 4A), a known inhibitor of let-7biogenesis (Viswanathan S R and Daley G Q. Cell. 140(4):445 (2010))resulted in a profound reduction of let-7b and 7c expression and anincrease in TGFβR1 and smooth muscle expression (FIGS. 4B-4F).Similarly, viral transduction of a let-7 “sponge” (Table 7; FIG. 4E)into EC led to a marked increase in TGFβR1 expression and appearance ofEndo-MT markers expression (FIGS. 4H-4K).

TABLE 7 sequences in let-7 sponge decoy let-7 family Sequence (5′→3′)hsa- UGAGGUAGUAG SEQ ID let-7a GUUGUAUAGUU NO: 90 hsa- UGAGGUAGUAGSEQ ID let-7b GUUGUGUGGUU NO: 91 hsa- UGAGGUAGUAG SEQ ID let-7cGUUGUAUGGUU NO: 92 hsa- UGAGGUAGUAG SEQ ID let-7d GUUGCAUAGU NO: 93 hsa-UGAGGUAGGAG SEQ ID let-7e GUUGUAUAGU NO: 94 hsa- UGAGGUAGUAG SEQ IDlet-7f AUUGUAUAGUU NO: 95 hsa- UGAGGUAGUAG SEQ ID let-7g UUUGUACAGUNO: 96 hsa- UGAGGUAGUAG SEQ ID let-7i UUUGUGCUGU NO: 97 mir-98UGAGGUAGUAA SEQ ID GUUGUAUUGUU NO: 98

To determine the in vivo relevance of let-7 inhibition in the regulationof TGFβRI, let-7b and let-7c miRNAs were directly targeted in mice usingcholesterol formulated antagomirs for improved stability and endothelialcell delivery. A single tail vein injection of 2 mg/kg ofcholesterol-formulated let-7b/c antagomirs did not produce anyalterations in mice's overall health, body weight or food intake. Sixdays later the animals were sacrificed and let-7 family expression wasdetermined in freshly isolated primary liver EC. There was a significantreduction in expression of let-7b and let-7c miRNA in EC fromantagomirs-treated compared to control mice (FIG. 4L). At the same time,there was no change in expression of other let-7 family members (FIG.4M). This reduction in let-7b/c levels was confirmed by increasedexpression of known let-7 target genes HMGA2, CDK6, and CDC25A (FIG.4N). Finally, let-7b/c antagomirs-treated mice EC demonstrated asignificant increase in TGFβRI and vimentin expression (FIG. 4N).

While reduction in Let-7b/c expression induced Endo-MT, expression ofthese miRNAs in primary EC in vitro following FRS2 knockdown (FIG. 5A)fully inhibited TGFβR1 and SMAD2 expression and prevented activation ofTGFβ signaling. As a result, expression of all SMC and mesenchymalmarkers declined to pre-FRS2 knockdown levels (FIGS. 5B-5I).

To gain an insight into the molecular mechanism by which FGF controlslet-7 expression, it was next determined whether FGF stimulation ofnormal EC affects its levels. FGF2 treatment of EC resulted in a timedependent increase in expression of all let-7 family miRNAs (FIGS.6A-6G). Since FGFR1/FRS2 complex primarily activates the ERK1/2 pathway(Gotoh N, et al. Proc Natl Acad Sci USA. 101(49):17144 (2004)), the roleof ERK1/2 in regulation of let-7 expression was investigated. Treatmentof EC with a MEK inhibitor U0126 led to a decrease in let-7 expression(FIGS. 6H-6M). To determine if ERK activation can restore let-7expression in EC with suppressed FGF signaling, EC subjected to FRS2knockdown were transduced with an adenoviral vector encoding aconstitutively active ERK (Ad-ME-LA) (Robinson M J, et al. Curr Biol.8(21):1141 (1998)) or LacZ control. Transduction of Ad-ME-LA, but not ofLacZ, increased let-7 expression, suppressed activation of TGFβsignaling including inhibition of Smad2 phosphorylation, and inhibitedsmooth muscle genes expression (FIG. 6N-6Q), indicating that regulationof TGFβ signaling by FGF is mediated by activation of theErk1/2-dependent signaling mechanism.

Example 4: TGFβ-Mediated Endo-MT Plays a Role in Vascular Graft Stenosis

Materials and Methods

Scaffold Fabrication & TEVG Implantation

Scaffolds were constructed from a nonwoven polyglycolic acid (PGA) mesh(Concordia Fibers) and a co-polymer sealant solution of poly-L-lactideand -ε-caprolactone (P(CL/LA)) as previously described (Roh J D, et al.Biomaterials. 29(10):1454 (2008)). For scaffold seeding studies, bonemarrow was collected from the femurs of syngeneic C57BL/6 mice.Following purification of the mononuclear cell component usingHistopaque-1086 (Sigma) centrifugation, one million mononuclear cellswere manually pipetted onto the scaffold. The seeded scaffold wasincubated in RPMI 1640 Medium (Gibco) overnight prior to implantation aspreviously described (Roh J D, et al. Biomaterials. 29(10):1454 (2008)).TEVG scaffolds were inserted into the infrarenal inferior vena cava(IVC) of 8-10 week old, female mice as previously described (Rob J D, etal. Biomaterials. 29(10):1454 (2008)).

In Vivo Microcomputed Tomography

Micro-CT imaging was performed using wild-type CB 17 mice at 10 weekspostoperatively as previously described using micro-CT scanner (exploreCT120; GE Healthcare, USA).

sFGFR1-IIIc Adenovirus Administration

sFGFR1-IIIc adenovirus was previously described (Murakami M, et al. JClin Invest. 118(10):3355 (2008)) at a dose of 5×10¹⁰ viral particlesper mouse 1 week prior to TEVG implantation by tail vein injection.Control mice were given equivalent volumes of sterile PBS. Serum levelof sFGFR1-IIIc was measured by a Human IgG Subclass Profile kit(Invitrogen).

Mouse Treatment with SB431542

Mice treated with TGFβRI kinase inhibitor were treated with SB431542hydrate (Sigma) in DMSO given by intraperitoneal injection twice a dayfrom post-operative day 0 to post-operative day 14 at a dose of 10mg/kg. Control mice were treated with equivalent volumes of sterileDMSO.

TEVG Analysis

Explanted grafts were pressure fixed in 10% formalin overnight and thenembedded in paraffin or glycolmethacrylate using previously publishedmethods (Roh J D, et al. Proc Natl Acad Sci USA. 107(10):4669 (2010)).Sections were stained with H&E or Gomori Trichrome. Graft luminaldiameters were measured using ImageJ software. Stenosis was defined asgreater than 50% decrease in luminal diameter. Critical stenosis wasdefined as 80% narrowing of the luminal diameter. Graft occlusion wasdefined as 100% narrowing of the luminal diameter. For EM studiesscaffolds were cut into 0.5 mm thick cross-sections and imaged on a FBIXL-30 scanning electron microscope (Hillsboro) as previously described(Roh J D, et al. Biomaterials. 29(10):1454 (2008)). TGFβ positive cellarea was measured using ImageJ software. Two separate sections of eachexplant were counterstained with hematoxalin and imaged at 400×magnification. The number of nuclei was then counted in five regions ofeach section and averaged. LacZ/SMA colocalized cells were quantified inthe same manner using double immunofluorescent staining imaged under 60×confocal magnification.

Tamoxifen Administration

100 mg tamoxifen (Sigma) was dissolved in 5 ml corn oil (20 mg/ml finalconcentration). The solution was mixed at 37° C. overnight. Pups werepipette fed with 0.05 mg/g tamoxifen solution every other day for 8times.

Whole Mount X-Gal Staining

The expression of LacZ in scaffolds was detected by X-gal(β-glactosidase) staining using beta-gal staining kit according to themanufacturer's instructions (MILLIPORE). Following X-gal staining, thescaffolds were refixed, dehydrated, embedded in paraffin, and sectionedat 6 μm. The paraffin sections were then countered with eosin beforebeing photographed.

Results

A tissue-engineered vascular graft (TEVG) stenosis model (Roh J D, etal. Biomaterials. 29(10):1454 (2008)) was used to examine whetherEndo-MT plays a role in vivo. Following implantation into the inferiorvena cava in an infrarenal position, TEVG develops extensive neointimacomposed of SMC and extracellular matrix leading to severe graftstenosis. Immunocytochemistry and qPCR analysis shows extensivedeposition of TGFβ and increased TGFβ1, TGFβR1 and vimentin expression(FIGS. 7A-7C).

To determine if transcriptional regulation of TGFβR1 by FGF signalingplays a role in TEVG stenosis, a soluble FGF trap (sFGFR1-IIIc), whichhas been previously demonstrated to virtually shutdown FGF signaling(Murakami M, et al. J Clin Invest. 118(10):3355 (2008)), wassystemically expressed one week before implantation of TEVG “seeded”with bone marrow mononuclear cells, a procedure known to dramaticallyimprove the graft patency (Roh J D, et al. Proc Natl Acad Sci USA.107(10):4669 (2010)). Two weeks after graft implantation, there weresignificantly higher TEVG neointima burden and stenosis rate in miceinjected with Ad-sFGFR1-IIIc compared to saline-injected control mice(FIG. 7D-7E).

To determine if Endo-MT contributes to this process, lineage analysis ofneointimal cells was performed following TEVG insertion into Rosa26 micecrossed with Tie2-Cre or Cdh5-CreERT2 mice with the latter Cre activatedby tamoxifen administration on day P2. In both cases LacZ-positive cellsare found throughout the entire neointima. Confocal analysis ofneointima samples double stained for LacZ and SM α-actin demonstratesco-localization of both markers in 40-50% of SMC (FIGS. 8A-8B).

Tie2-CreXRosa26 mice were treated with the TGFBR1 inhibitor SB-431542 at10 mg/kg twice daily by intraperitoneal administration for 2 weeksfollowing implantation of our TEVG (n=16). Matched control mice receivedintraperitoneal injection of sterile DMSO (n=25). Results of thesestudies showed that TGFBR1 inhibitor treatment significantly increasesTEVG luminal diameter and graft patency at 2 weeks in unneeded grafts(p<0.001 for diameter, p<0.01 for stenosis) (FIGS. 8C and 8D). Drugtreatment also allows for proper neotissue creation with an organizedCD31 positive endothelial cell layer lining an SMA positive smoothmuscle cell layer in contrast to the typical untreated control graftthat occludes as a result of accumulation of SMA positive smooth musclecells.

In order to unravel the mechanism by which SB-431542 treatment preventsTEVG stenosis, we treated mice from the Tie2 lineage-tracing model(n=10) with TGFBR1 inhibitor drug and then performed confocal analysisand cellular quantification of TEVG samples double stained for LacZ andSMA. Results of these studies showed that drug treatment improvespatency by reducing the occurrence of EMT, as demonstrated by asignificant reduction in LacZ-positive smooth muscle cells in occludedgrafts in drug treated mice (p<0.05) (FIG. 8E).

Example 5: Local Delivery of TGFBR1 Inhibitor Inhibits Stenosis WithoutCell Seeding and Maintains Normal Neovessel Formation

Materials and Methods

Microparticle Synthesis and Characterization

SB-431542 was encapsulated in avidin-coated PLGA microparticles using amodified oil/water single emulsion technique as previously described(Fahmy, T. M., et al. Biomaterials 26:5727-5736 (2005)). Controlmicroparticles were synthesized without SB-431542. Microparticle sizeand morphology were analyzed via scanning electron microscopy (SEM) aspreviously described (Rob, J. D., et al. Proc Natl Acad Sci USA107:4669-4674 (2010)).

Preparation of Adhesive Peptide Tether

Poly-L-lysine-LC-LC-biotin (pLLB) was synthesized and used as anadhesive peptide tether to enhance loading of PGA-P(CL/LA) scaffoldswith avidin-coated microparticles. 1.66 mg EZ-Link sulfoNHS-LC-LC-biotin was reacted with 10 ml of a 0.1 mg/ml solution ofpoly-L-lysine (MW 70,000-150,000, Sigma) in PBS for 2 hours at 4 C,dialyzed in PBS for 72 hours, and stored at 4 C.

Loading of TEVG Scaffolds with SB-431542-Eluting Microparticles

Nonspecific adsorption of avidin-coated PLGA microparticles toPGA-P(CL/LA) scaffolds not treated with pLLB was titrated by incubatingtrimmed scaffolds with 1 ml of 1, 5, or 10 mg/ml of microparticles inPBS for 10, 30 or 60 minutes. Particle-loaded TEVG scaffolds wereimmediately snap frozen in liquid nitrogen and lyophilized for 6 hoursbefore imaging. Scaffold loading efficiency was determined with ImageJsoftware from three SEM images per scaffold cross-section, innersurface, and outer surface by calculating the mean surface density ofparticles. The effect of scaffold pretreatment with pLLB on scaffoldloading efficiency was assessed from particle loading density as aboveafter PGA-P(CL/LA) scaffolds were incubated with 1 ml of 0.01, 0.1 or 1mg/ml pLLB for 60 minutes on a rotary shaker, washed with dH2O,incubated with 1 ml 5 mg/ml avidin-coated PLGA microparticles, washedwith dH₂O, snap frozen in liquid nitrogen, lyophilized for 6 hours, andimaged by SEM. For in vitro and in vivo studies, PGA-P(CL/LA) scaffoldswere incubated with pLLB for 30 minutes at 20 C, washed with dH2O,incubated with 5 mg/ml empty or SB-431542-eluting avidin-coated PLGAmicroparticles for 30 minutes, washed with dH2O, snap frozen in liquidnitrogen, and lyophilized for 6 hours before desiccated storage.

Characterization of SB-431542 Release from Microparticles and Scaffolds

Total encapsulation was approximated as the amount of SB-431542 releasedover a 14-day period. Percent encapsulation efficiency was calculated astotal encapsulation divided by maximum theoretical encapsulation. 5 mgof avidin-coated PLGA microparticles containing SB-431542, PGA-P(CL/LA)trimmed scaffolds and treated with pLLB and SB-431542-elutingmicroparticles as above, and SB-431542-eluting PGA-P(CL/LA) trimmedscaffolds were incubated with PBS in triplicate on a rotary shaker at37° C. Samples were removed at specific time points and centrifuged at13200 RPM for 10 minutes. 300 μl of supernatant was drawn and replacedwith 300 μl PBS. Concentration of SB-431542 in supernatant diluted with600 μl PBS was determined by spectrophotometry at 320 nm.

Bioactivity of Encapsulated SB-431542

SB-431542 was released into 1 ml PBS from 10 mg avidin-coated PLGAmicroparticles and one untrimmed SB-431542-eluting PGA-P(CL/LA) scaffoldin 2 ml microcentrifuge tubes at 37° C. At 48 hours, samples werecentrifuged at 13200 RPM for 10 minutes, supernatants were collected andanalyzed by spectrophotometry at 320 nm. SB-431542 concentrations wereadjusted to 10 μM by dilution with PBS. 3T3 human fibroblasts wereplated at 500,000/well and stimulated at confluence with 700 ul 10 μMSB-431542 in PBS eluted from particles or scaffolds, a stock solution of10 or 1 μM SB-431542 containing <1% DMSO, or PBS. After 30 minutes at37° C., cells were washed and stimulated with 200 ul 2 ng/ml recombinanthuman TGF-β1 (BD Biosciences) for 1 hour at 37 C. Cells were lysed andprotein samples were separated by gel electrophoresis with a 12%polyacrylamide gel and probed with primary antibody to phosphorylatedSMAD-2 (ser426/ser428, Cell Signaling Technology) and secondary goatanti-rabbit IgG (Cell Signaling Technology). The gel was stripped andreprobed with anti-SMAD2/3 as a loading control.

Results

A microparticle system was developed for local delivery of the TGFBR1inhibitor SB-431542 in order to minimize possible effects of systemicdelivery. This system was characterized to show that there is steadyrelease of the drug across the full 2-week time course during which thegrafts are implanted (FIGS. 14A and 14B), and showed that the releaseddrug maintains its biologic activity. A simpler local drug deliverysystem was also developed by which the TGFBR1 inhibitor was added to thesolvent used to make the grafts. This system also demonstrated afavorable release profile and continued biologic activity of thereleased drug (FIGS. 14A and 14B). Both types of drug-eluting graftswere then implanted in the mouse model (n=10 for drug in microparticles,n=24 for drug in solvent) and their patency compared to grafts withempty microparticles (n=10) or control grafts (n=25). Results of thesestudies showed that local drug delivery significantly increases patencyat 2 weeks in unseeded grafts (p<0.01) and also enabled neotissuecreation (FIGS. 14C and 14D).

We claim:
 1. A cell-free polymeric vascular graft, scaffold, or stentcomprising an effective amount of a TGFβ inhibitor, a SMAD2 inhibitor,an FGF receptor agonist, a Let-7 agonist, or a combination thereof, toincrease the recruitment of host cells to the graft, scaffold, or stent,and promote endothelial cell growth but not smooth muscle cellproliferation, relative to the graft, scaffold, or stent without theTGFβ inhibitor, a SMAD2 inhibitor, an FGF receptor agonist, a Let-7agonist, or a combination thereof.
 2. The cell-free polymeric vasculargraft, scaffold, or stent of claim 1, comprising biodegradable orbioabsorbable polymers.
 3. The cell-free polymeric vascular graft,scaffold, or stent of claim 2, wherein the biodegradable orbioabsorbable polymers are selected from the group consisting ofpoly(lactic acid), poly(glycolic acid), polyanhydrides,poly(ortho)esters, polyesters, polyurethanes, poly(butic acid),poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), andpoly(lactide-co-caprolactone), or combinations, blends or co-polymersthereof.
 4. The cell-free polymeric vascular graft, scaffold, or stentof claim 2, wherein the biodegradable or bioabsorbable polymers areformed into a fiber-based mesh.
 5. The cell-free polymeric vasculargraft, scaffold, or stent of claim 4 wherein the fiber-based mesh is anon-woven mesh.
 6. The cell-free polymeric vascular graft, scaffold, orstent of claim 2, wherein the vascular graft further comprises apolymeric sealant.
 7. The cell-free polymeric vascular graft, scaffold,or stent of claim 6, wherein the polymeric sealant comprises aco-polymer of ε-caprolactone and L-lactide.
 8. The cell-free polymericvascular graft, scaffold, or stent of claim 1, wherein the TGFβinhibitor or SMAD2 inhibitor is selected from the group consisting of4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide(SB431542), 2-(5-Chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine(SB208),6-[2-(1,1-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline(SB525334),4-(5-(benzo[d][1,3]dioxol-5-yl)-4-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)4yridin[2.2.2]octane-1-carboxamide(SM16),4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)4yridine4-6-carboxamide(LY2157299),3-((5-([1,2,4]triazolo[1,5-a]4yridine-6-yl)-4-(6-methylpyridin-2-yl)thiazol-2-ylamino)methyl)benzonitrile(EW-7203), and(2-butyl-4-chloro-1-{[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-1H-imidazol-5-yl)methanol(Losartan).
 9. The cell-free polymeric vascular graft, scaffold, orstent of claim 1, wherein the Let-7 agonist is a polynucleotide encodinga Let-7 pri-miRNA, pre-miRNA, mature miRNA, or RNAi effective insilencing TGFβRI gene expression.
 10. The cell-free polymeric vasculargraft, scaffold, or stent of claim 9, wherein the Let-7 agonistcomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NO:102, 103, 104, 105, 105, 106, 107, 108, 109, 110, 111, 112, or113, or a fragment or variant thereof capable of binding a let-7 targetsequence and silence TGFβRI gene expression.
 11. The cell-free polymericvascular graft, scaffold, or stent of claim 1, wherein the vasculargraft, scaffold, or stent further comprises an active agent selectedfrom the group consisting of anti-thrombogenic agents,anti-proliferative agents, anti-inflammatory agents, antiproliferativeagents, anesthetic agents, anti-coagulants, cholesterol-lowering agents,vasodilating agents, and agents which interfere with endogenousvascoactive mechanisms.
 12. The cell-free polymeric vascular graft,scaffold, or stent of claim 1, wherein the TGFβ inhibitor, SMAD2inhibitor, FGF receptor agonist, Let-7 agonist, or combination thereof,is dispersed throughout the vascular graft, scaffold, or stent.
 13. Thecell-free polymeric vascular graft, scaffold, or stent of claim 1,wherein the TGFβ inhibitor, SMAD2 inhibitor, FGF receptor agonist, Let-7agonist, or combination thereof, is encapsulated in the form ofmicrospheres, nano spheres, microparticles and/or microcapsules that areseeded into the vascular graft, scaffold, or stent.
 14. The cell-freepolymeric vascular graft, scaffold, or stent of claim 1, wherein theTGFβ inhibitor or the SMAD2 inhibitor comprises an antibody, an antibodyfragment, an antigen-binding variable domain of an antibody, a solublereceptor, or a mutant ligand.
 15. The cell-free polymeric vasculargraft, scaffold, or stent of claim 1, wherein the effective amount theTGFβ inhibitor, the SMAD2 inhibitor, the FGF receptor agonist, the Let-7agonist, or the combination thereof, is an amount effective to inhibitendothelial to mesenchymal transition of vascular endothelial cells atthe site of the vascular graft, scaffold, or stent implantation.
 16. Thecell-free polymeric vascular graft, scaffold, or stent of claim 1,wherein the cell-free polymeric vascular graft, scaffold, or stentretains its patency for at least about two weeks following implantation.